DNA molecules from maize and methods of use thereof

ABSTRACT

The present invention relates to DNA polynucleotides for regulating gene expression in plants. In particular, the invention relates to 5′ regulatory sequences isolated from  Zea mays  that are useful for regulating gene expression of heterologous DNA molecules in plant roots. The invention also relates to transgenic plants containing the heterologous DNA molecules.

[0001] This application claims priority to an earlier filed U.S.Provisional application No. 60/310,370, filed Aug. 6, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to the isolation and use of nucleicacid molecules for control of gene expression in plants. Specifically anovel DNA molecule isolated from Zea mays and its use to expresstransgene products in plants. The methods of the present inventionprovide for the isolation of novel DNA molecules that have promoterfunction for enhanced expression in root tissues of transgenic plants.

BACKGROUND OF THE INVENTION

[0003] One of the goals of plant genetic engineering is to produceplants with agronomically important characteristics or traits. Recentadvances in genetic engineering have provided the requisite tools totransform plants to contain and express foreign genes (Kahl et al.(1995) World Journal of Microbiology and Biotechnology 11:449-460).Particularly desirable traits or qualities of interest for plant geneticengineering would include, but are not limited to, resistance to insectsand other pests and disease-causing agents, tolerances to herbicides,enhanced stability, yield, or shelf-life, environmental tolerances, andnutritional enhancements. The technological advances in planttransformation and regeneration have enabled researchers to take piecesof DNA, such as a gene or genes from a heterologous source, or a nativesource, but modified to have different or improved qualities, andincorporate the exogenous DNA into the plant's genome. The gene orgene(s) can then be expressed in the plant cell to exhibit the addedcharacteristic(s) or trait(s). In one approach, expression of a novelgene that is not normally expressed in a particular plant or planttissue may confer a desired phenotypic effect. In another approach,transcription of a gene or part of a gene in an antisense orientationmay produce a desirable effect by preventing or inhibiting expression ofan endogenous gene.

[0004] Isolated plant promoters are useful for modifying plants throughgenetic engineering to have desired phenotypic characteristics. In orderto produce such a transgenic plant, a vector that includes aheterologous gene sequence that confers the desired phenotype whenexpressed in the plant is introduced into the plant cell. The vectoralso includes a plant promoter that is operably linked to theheterologous gene sequence, often a promoter not normally associatedwith the heterologous gene. The vector is then introduced into a plantcell to produce a transformed plant cell, and the transformed plant cellis regenerated into a transgenic plant. The promoter controls expressionof the introduced DNA sequence to which the promoter is operably linkedand thus affects the desired characteristic conferred by the DNAsequence.

[0005] Since the promoter is a 5′ regulatory element that plays anintegral part in the overall expression of a gene or gene(s), it wouldbe advantageous to have a variety of promoters to tailor gene expressionsuch that a gene or gene(s) is transcribed efficiently at the right timeduring plant growth and development, in the optimal location in theplant, and in the amount necessary to produce the desired effect. In onecase, for example, constitutive expression of a gene product may bebeneficial in one location of the plant, but less beneficial in anotherpart of the plant. In other cases, it may be beneficial to have a geneproduct produced at a certain developmental stage of the plant, or inresponse to certain environmental or chemical stimuli, or in aparticular plant tissue or organ. The commercial development ofgenetically improved germplasm has also advanced to the stage ofintroducing multiple traits into crop plants, often referred to as agene stacking approach. In this approach, multiple genes conferringdifferent characteristics of interest can be introduced into a plant. Itis important when introducing multiple genes into a plant, that eachgene is modulated or controlled for optimal expression and that theregulatory elements are diverse, to reduce the potential of genesilencing that can be caused by recombination of homologous sequences.In light of these and other considerations, it is apparent that optimalcontrol of gene expression and regulatory element diversity areimportant in plant biotechnology.

[0006] The proper regulatory sequences must be present and in the properlocation with respect to the DNA sequence of interest, for the newlyinserted DNA to be transcribed and thereby, if desired, translated intoa protein in the plant cell. These regulatory sequences include but arenot limited to a promoter, a 5′ untranslated leader, and a 3′polyadenylation sequence. The ability to select the tissues in which totranscribe such foreign DNA, and the time during plant growth in whichto obtain transcription of such foreign DNA is also possible through thechoice of appropriate promoter sequences that control transcription ofthese genes.

[0007] A variety of different types or classes of promoters can be usedfor plant genetic engineering. Promoters can be classified on the basisof range or tissue specificity. For example, promoters referred to asconstitutive promoters are capable of transcribing operatively linkedDNA sequences efficiently and expressing said DNA sequences in multipletissues. Tissue-enhanced or tissue-specific promoters can be foundupstream and operatively linked to DNA sequences normally transcribed inhigher levels in certain plant tissues or specifically in certain planttissues. This can be referred to as differential expression whenexpression levels are compared relative to other plant cells or tissues.Other classes of promoters would include but are not limited toinducible promoters that can be triggered by external stimuli such aschemical agents, developmental stimuli, or environmental stimuli. Thus,the different types of promoters desired can be obtained by isolatingthe upstream 5′ regulatory regions of DNA sequences that are transcribedand expressed in a constitutive, tissue-enhanced, or inducible manner.

[0008] The technological advances of high-throughput sequencing andbioinformatics has provided additional molecular tools for promoterdiscovery. Particular target plant cells, tissues, or organs at aspecific stage of development, or under particular chemical,environmental, or physiological conditions can be used as sourcematerial to isolate the mRNA and construct cDNA libraries. The cDNAlibraries are quickly sequenced and the expressed sequences cataloguedelectronically. Using sequence analysis software, thousands of sequencescan be analyzed in a short period, and sequences from selected cDNAlibraries can be compared. The combination of laboratory andcomputer-based subtraction methods allows researchers to scan andcompare cDNA libraries and identify sequences with a desired expressionprofile. For example, sequences expressed preferentially in one tissuecan be identified by comparing a cDNA library from one tissue to cDNAlibraries of other tissues and electronically “subtracting” commonsequences to find sequences only expressed in the target tissue ofinterest. The tissue enhanced sequence can then be used as a probe orprimer to clone the corresponding full-length cDNA. A genomic library ofthe target plant can then be used to isolate the corresponding gene andthe associated regulatory elements, including promoter sequences.

[0009] Multiple promoter sequences that confer a desired expressionprofile such as promoters capable of regulating expression of operablylinked genes in multiple tissues can be isolated by selectivelycomparing cDNA libraries of target tissues of interest with non-targetor background cDNA libraries to find the 5′ regulatory regionsassociated with the expressed sequences in those target libraries. Theisolated promoter sequences can be used for selectively modulatingexpression of any operatively linked gene and provide additionalregulatory element diversity in a plant expression vector in genestacking approaches.

SUMMARY OF THE INVENTION

[0010] The present invention provides nucleic acid molecules comprisinga DNA polynucleotide sequence set forth in SEQ ID NO: 3 or anyfragments, regions, cis elements or homologue of the sequence that arecapable of regulating transcription of operably linked DNA sequences.

[0011] Another aspect of the present invention relates to the use of atleast one fragment, region, or cis element thereof of SEQ ID NO: 3 thatcan be combined to create novel promoter DNA sequences or used in anovel combination with another heterologous regulatory sequence tocreate a hybrid or chimeric promoter capable of modulating transcriptionof an operably linked DNA sequence.

[0012] Hence, the present invention relates to the use of a DNApolynucleotide disclosed in SEQ ID NO: 3, or any fragment, region, ciselements or homologue of the disclosed molecules that are capable ofregulating transcription of an operably linked DNA sequence. Therefore,the invention not only encompasses the DNA polynucleotide as disclosedin SEQ ID NO: 3, but also includes any truncated or deletionderivatives, or fragments or regions thereof that are capable offunctioning independently as a plant promoter including cis elementsthat are capable of functioning as regulatory sequences in conjunctionwith one or more regulatory sequences when operably linked to atranscribable sequence.

[0013] The present invention thus encompasses a novel plant promoter ora hybrid or chimeric promoter that functions in plants to cause RNAtranscription comprising a DNA polynucleotide of SEQ ID NO 3. The hybridor chimeric promoters can consist of any length fragments, regions, orcis elements of the disclosed DNA polynucleotide of SEQ ID NO: 3combined with any other transcriptionally active minimal or full-lengthpromoter. For example, a fragment of the DNA polynucleotide set forth inSEQ ID NO: 3 may be combined with a plant DNA virus promoter or otherpromoter sequences or cis elements identified therein to construct anovel hybrid promoter. More preferably, the DNA polynucleotide set forthin SEQ IN NO: 3 or fragments thereof, may be combined with promoters orfragments of promoters that function to direct enhanced transcription ofan operably linked DNA molecule into plant root cells and tissues.

[0014] A DNA polynucleotide fragment of SEQ ID NO: 3 from position110-192 and DNA polynucleotide fragments at least 90% homologous to thissequence is an aspect of the present invention. A DNA polynucleotidefragment of SEQ ID NO: 3 from position 126-164 and DNA polynucleotidefragments at least 90% homologous to this sequence is an aspect of thepresent invention.

[0015] The present invention also encompasses DNA molecules thatcomprise plant expression constructs containing the disclosed DNApolynucleotide set forth in SEQ ID NO: 3 or any fragments, regions, ciselements or homologue thereof, including novel promoters generated usingthe disclosed DNA sequences or any fragment, region, or cis element ofthe disclosed DNA sequences operably linked to a heterologous DNApolynucleotide comprising a gene of interest, operably linked to a 3′termination polynucleotide sequence.

[0016] The present invention also includes any transgenic plant cellsand transgenic plants containing the DNA molecule comprising a plantexpression construct containing the DNA polynucleotide set forth in SEQID NO: 3, or any fragments, regions, cis elements or homologue thereofin operable linkage to a heterologous DNA polynucleotide sequence.

[0017] The present invention also provides a DNA molecule for enhancingtranscription of a linked DNA molecule preferably in plant root cellsand root tissues by linking a heterologous gene of interest DNA moleculeto the DNA polynucleotide set forth in SEQ ID NO: 3 or any fragment,region, cis element or homologue thereof, and a 3′ terminationpolynucleotide sequence

[0018] The present invention also provides a transgenic plant made by amethod comprising: a) constructing a plant expression constructcomprising (i) a DNA polynucleotide comprising the DNA sequence setforth in SEQ ID NO: 3, or fragments, regions, cis elements or homologuethereof, operably linked to, (ii) a transcribable heterologous DNAsequence and (iii) a 3′ termination polynucleotide sequence, b)transforming a plant cell with the plant expression construct, and c)regenerating the transformed plant cell into a fertile plant.

[0019] The DNA polynucleotide set forth in SEQ ID NO: 3 or fragmentsthereof, can be used in a method of isolating DNA polynucleotides,wherein SEQ ID NO: 3 or fragments thereof can be used as probes orprimer molecules to isolate similar DNA polynucleotides that function aspromoters to enhance transcription of a linked gene of interest in plantroot cells or root tissues.

[0020] The DNA polynucleotide set forth in SEQ ID NO: 12 or fragmentsthereof, can be used in a method of isolating DNA polynucleotides,wherein SEQ ID NO: 12 or fragments thereof can be used as probes orprimer molecules to isolate associated DNA polynucleotides that functionas promoters to enhance transcription of a linked gene of interest inplant root cells or root tissues. The DNA polynucleotides isolated bythe method, wherein the DNA polynucleotides are isolated from monocotplants and function as promoters that enhance expression of aheterologous gene sequence in plant root cells and root tissues.

[0021] A DNA promoter polynucleotide can be isolated by a methodcomprising the steps of: (i) preparation of plant genomic DNA; and (ii)preparation of a mixture of degenerate DNA polynucleotide primers to alength of the translation product of SEQ ID NO: 12; and (iii) mixingsaid degenerate DNA polynucleotide primers and genomic DNA with a secondDNA polynucleotide primer not homologous to SEQ ID NO: 12; and (iv)subjecting said mixture to a PCR condition that provides an amplicon;and (v) purifying said amplicon; (vi) inserting said amplicon into anexpression construct; and (vii) testing said expression construct forexpression of a reporter molecule.

[0022] The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a plasmid map of pMON19469

[0024]FIG. 2 is a plasmid map of pMON33336

[0025]FIG. 3 is a plasmid map of pMON33292

[0026]FIG. 4 is a plasmid map of pMON33333

[0027]FIG. 5 is a plasmid map of pMON18365

[0028]FIG. 6 is a plasmid map of pMON46152

[0029]FIG. 7 is a plasmid map of pMON46154

[0030]FIG. 8 is a plasmid map of pMON46155

[0031]FIG. 9 is a graph of the reporter gene (GUS) expression analysisperformed on young leaves (newly emerged), fourth surviving leaf frombase of the plant, and root tissues of R₀ corn plants transformed withcontrol constructs and pMON46152 containing the root enhanced promoterof the present invention.

[0032]FIG. 10 is a graph of the reporter gene (GUS) expression analysisperformed on six tissues of R₁ corn plants transformed with constructspMON18365, pMON46152, pMON46154, and pMON46155.

[0033]FIG. 11 is a plasmid map of pMON64106

[0034]FIG. 12 is a plasmid map of pMON64107

[0035]FIG. 13 is a plasmid map of pMON64108

[0036]FIG. 14 is a plasmid map of pMON64109

DETAILED DESCRIPTION OF THE INVENTION

[0037] Definitions and Methods

[0038] The following definitions and methods are provided to betterdefine the present invention and to guide those of ordinary skill in theart in the practice of the present invention. Unless otherwise noted,terms are to be understood according to conventional usage by those ofordinary skill in the relevant art.. The nomenclature for DNA bases asset forth at 37 CFR §1.822 is used. The standard one- and three-letternomenclature for amino acid residues is used.

[0039] “Nucleic acid (sequence)” or “polynucleotide (sequence)” refersto single- or double-stranded DNA or RNA of genomic or synthetic origin,i.e., a polymer of deoxyribonucleotide or ribonucleotide bases,respectively, read from the 5′ (upstream) end to the 3′ (downstream)end. The nucleic acid can represent the sense or complementary(antisense) strand.

[0040] “Native” refers to a naturally occurring (“wild-type”) nucleicacid sequence.

[0041] “Heterologous” refers to a DNA sequence that originates from adifferent species. Heterologous can also mean being different from whereit exists in nature, such as from a different place in the genome as ina different member of a gene family, or a different allele of the samegene or if from the same source, is modified from its original form.

[0042] An “isolated” nucleic acid sequence or isolated DNApolynucleotide is substantially separated or purified away from othernucleic acid sequences with that the nucleic acid is normally associatedin the cell of the organism in which the nucleic acid naturally occurs,i.e., other chromosomal or extrachromosomal DNA. The term embracesnucleic acids and polynucleotides that are biochemically purified so asto substantially remove contaminating nucleic acids and other cellularcomponents. The term also embraces recombinant nucleic acids andchemically synthesized polynucleotides.

[0043] The term “substantially purified, as used herein, refers to amolecule separated from substantially all other molecules normallyassociated with it in its native state. More preferably, a substantiallypurified molecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free from the other molecules (exclusiveof solvent) present in the natural mixture. The term “substantiallypurified” does not encompass molecules present in their native state.

[0044] A first nucleic acid sequence displays “substantial identity” toa reference nucleic acid sequence if, when optimally aligned (withappropriate nucleotide insertions or deletions totaling less than 20percent of the reference sequence over the window of comparison) withthe other nucleic acid (or its complementary strand), there is at leastabout 75% nucleotide sequence identity, preferably at least about 80%identity, more preferably at least about 85% identity, and mostpreferably at least about 90% identity over a comparison window of atleast 20 nucleotide positions, preferably at least 50 nucleotidepositions, more preferably at least 100 nucleotide positions, and mostpreferably over the entire length of the first nucleic acid. Optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith and Waterman Adv. Appl. Math.2:482, 1981; by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;preferably by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis. Thereference nucleic acid may be a full-length molecule or a portion of alonger molecule. Alternatively, two nucleic acids are have substantialidentity if one hybridizes to the other under stringent conditions, asdefined below.

[0045] A first polynucleotide sequence is “operably linked” with asecond polynucleotide sequence when the sequences are so arranged thatthe first polynucleotide sequence affects the function of the secondpolynucleotide sequence. Preferably, the two sequences are part of asingle contiguous nucleic acid molecule and more preferably areadjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the genein a cell.

[0046] A “recombinant” nucleic acid is made by an artificial combinationof two otherwise separated segments of sequence, e.g., by chemicalsynthesis or by the manipulation of isolated segments of nucleic acidsby genetic engineering techniques. Techniques for nucleic-acidmanipulation are well-known (see, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989, herein referred to as Sambrook et al., 1989,and Ausubel et al., 1992).

[0047] Methods for chemical synthesis of nucleic acids are discussed,for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862,1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers.

[0048] An “artificial polynucleotide sequence” can be designed andchemically synthesized for enhanced expression in particular host cellsand for the purposes of cloning into appropriate vectors. Computerprograms are available for these purposes including, but not limited tothe “BestFit” or “Gap” programs of the Sequence Analysis SoftwarePackage, Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis. 53711.

[0049] “Amplification” of nucleic acids or “nucleic acid reproduction”refers to the production of additional copies of a nucleic acid sequenceand is carried out using polymerase chain reaction (PCR) technologies. Avariety of amplification methods are known in the art and are described,inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCRProtocols: A Guide to Methods and Applications, ed. Innis et al.,Academic Press, San Diego, 1990. In PCR, a primer refers to a shortoligonucleotide of defined sequence that is annealed to a DNA templateto initiate the polymerase chain reaction.

[0050] “Transformed”, “transfected”, or “transgenic” refers to a cell,tissue, organ, or organism into which has been introduced a foreignnucleic acid, such as a recombinant vector. Preferably, the introducednucleic acid is integrated into the genomic DNA of the recipient cell,tissue, organ or organism such that the introduced nucleic acid isinherited by subsequent progeny. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing such a “transgenic” plant asa parent in a cross and exhibiting an altered phenotype resulting fromthe presence of a recombinant construct or vector.

[0051] The term “gene” refers to chromosomal DNA, plasmid DNA, cDNA,synthetic DNA, or other DNA that encodes a peptide, polypeptide,protein, or RNA molecule, and regions flanking the coding sequenceinvolved in the regulation of expression. Some genes can be transcribedinto messenger RNA (mRNA) then translated into polypeptides (structuralgenes); other genes can be transcribed into other types of RNA (e.g.rRNA, tRNA,. antisense RNA); and these other types of RNAs can functionas regulators of expression.

[0052] “Expression” of a gene refers to the transcription of a gene toproduce the corresponding mRNA. This mRNA may be translated to producethe corresponding gene product, i.e., a peptide, polypeptide, orprotein. Gene expression is controlled or modulated by regulatoryelements including 5′ regulatory elements such as promoters.

[0053] “Genetic component” refers to any nucleic acid sequence orgenetic element that may also be a component or part of an expressionvector. Examples of genetic components include, but are not limited topromoter regions, 5′ untranslated leaders, introns, genes, 3′untranslated regions, and other regulatory sequences or sequences thataffect transcription or translation of one or more nucleic acidsequences.

[0054] The terms “recombinant DNA construct”, “recombinant vector”,“expression vector” or “expression cassette” refer to any agent such asa plasmid, cosmid, virus, BAC (bacterial artificial chromosome),autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence,derived from any source, capable of genomic integration or autonomousreplication, comprising a DNA molecule in which one or more DNAsequences have been linked in a functionally operative manner.Bacterial, fungal, plant, animal are often used to describe theconstruct or vector, for example, “plant expression cassette” includesall of the genetic elements known to those skilled in the art of plantmolecular biology that permit the expression product to be produced inplant cells.

[0055] “Complementary” refers to the natural association of nucleic acidsequences by base-pairing (A-G-T pairs with the complementary sequenceT-C-A). Complementarity between two single-stranded molecules may bepartial, if only some of the nucleic acids pair are complementary; orcomplete, if all bases pair are complementary. The degree ofcomplementarity affects the efficiency and strength of hybridization andamplification reactions.

[0056] “Homology” refers to the level of similarity between nucleic acidor amino acid sequences in terms of percent nucleotide or amino acidpositional identity, respectively, i.e., sequence similarity oridentity. Homology also refers to the concept of similar functionalproperties among different nucleic acids or proteins, e.g., promotersthat have similar function may have homologous cis elements.Polynucleotides are homologous when under certain conditions theyspecifically hybridize to form a duplex molecule. A DNA molecule canhave substantial identity with another DNA molecule, if the sequence ofpolynucleotides is homologous or complementary under specifiedconditions. Under these specified conditions, referred to as stringencyconditions, one DNA molecule can be used as a probe or primer toidentify other DNA molecules that share homology. Homology can also bedetermined by computer programs that align polynucleotide sequences andestimate the ability of DNA molecules to form duplex molecules undercertain stringency conditions. A length of polynucleotide sequence canbe related to another polynucleotide sequence by the number of identicalnucleotides of the length, this is referred to as percent homology. DNAmolecules from different sources that share a high degree of homologyare referred to as “homologues”.

[0057] “ESTs” or Expressed Sequence Tags are short sequences of randomlyselected clones from a cDNA (or complementary DNA) library that arerepresentative of the cDNA inserts of these randomly selected clones(McCombie, et al., Nature Genetics, 1:124, 1992; Kurata, et al., NatureGenetics, 8: 365,1994; Okubo, et al., Nature Genetics, 2: 173, 1992).

[0058] The term “electronic Northern” refers to a computer-basedsequence analysis that allows sequences from multiple cDNA libraries tobe compared electronically based on parameters the researcher identifiesincluding abundance in EST populations in multiple cDNA libraries, orexclusively to EST sets from one or combinations of libraries.

[0059] “Subsetting” refers to any method of comparing nucleic acidsequences from different or multiple sources that can be used toidentify the profile of the nucleic acid sequences that reflects genetranscription activity and message stability in a particular tissue, ata particular time, or under particular conditions.

[0060] “Promoter” refers to a nucleic acid sequence located upstream or5′ to a translational start codon of an open reading frame (orprotein-coding region) of a gene and that is involved in recognition andbinding of RNA polymerase II and other proteins (trans-actingtranscription factors) to initiate transcription. A “plant promoter” isa native or non-native promoter that is functional in plant cells.Constitutive promoters are functional in most or all tissues of a plantthroughout plant development. Tissue-, organ- or cell-specific promotersare expressed only or predominantly in a particular tissue, organ, orcell type, respectively. Rather than being expressed “specifically” in agiven tissue, organ, or cell type, a promoter may display “enhanced”expression, i.e., a higher level of expression, in one part (e.g., celltype, tissue, or organ) of the plant compared to other parts of theplant. Temporally regulated promoters are functional only orpredominantly during certain periods of plant development or at certaintimes of day, as in the case of genes associated with circadian rhythm,for example. Inducible promoters selectively express an operably linkedDNA sequence in response to the presence of an endogenous or exogenousstimulus, for example by chemical compounds (chemical inducers) or inresponse to environmental, hormonal, chemical, and/or developmentalsignals. Inducible or regulated promoters include, for example,promoters regulated by light, heat, stress, flooding or drought,phytohormones, wounding, or chemicals such as ethanol, jasmonate,salicylic acid, or safeners.

[0061] Any plant promoter can be used as a 5′ regulatory sequence formodulation expression of a particular gene or genes. One preferredpromoter would be a plant RNA polymerase II promoter. Plant RNApolymerase II promoters, like those of other higher eukaryotes, havecomplex structures and are comprised of several distinct elements. Onesuch element is the TATA box or Goldberg-Hogness box, that is requiredfor correct expression of eukaryotic genes in vitro and accurate,efficient initiation of transcription in vivo. The TATA box is typicallypositioned at approximately −25 to −35, that is, at 25 to 35 basepairs(bp) upstream (5′) of the transcription initiation site, or cap site,which is defined as position +1 (Breathnach and Chambon, Ann. Rev.Biochem. 50:349-383, 1981; Messing et al., In: Genetic Engineering ofPlants, Kosuge et al., eds., pp. 211-227, 1983). Another common element,the CCAAT box, is located between −70 and −100 bp. In plants, the CCAATbox may have a different consensus sequence than the functionallyanalogous sequence of mammalian promoters (the plant analogue has beentermed the “AGGA box” to differentiate it from its animal counterpart;Messing et al., In: Genetic Engineering of Plants, Kosuge et al., eds.,pp. 211-227, 1983). In addition, virtually all promoters includeadditional upstream activating sequences or enhancers (Benoist andChambon, Nature 290:304-310, 1981; Gruss et al., Proc. Nat. Acad. Sci.USA 78:943-947, 1981; and Khoury and Gruss, Cell 27:313-314, 1983)extending from around −100 bp to −1,000 bp or more upstream of thetranscription initiation site.

[0062] When fused to heterologous DNA sequences, such promoterstypically cause the fused sequence to be transcribed in a manner that issimilar to that of the gene sequence with which the promoter is normallyassociated. Promoter fragments that include regulatory sequences can beadded (for example, fused to the 5′ end of, or inserted within, anactive promoter having its own partial or complete regulatory sequences(Fluhr et al., Science 232:1106-1112, 1986; Ellis et al., EMBO J.6:11-16, 1987; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Poulsen and Chua, Mol. Gen. Genet. 214:16-23, 1988;Comai et al., Plant Mol. Biol. 15:373-381, 1991). Alternatively,heterologous regulatory sequences can be added to the 5′ upstream regionof an inactive, truncated promoter, e.g., a promoter including only thecore TATA and, sometimes, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991).

[0063] Promoters are typically comprised of multiple distinct“cis-acting transcriptional regulatory elements,” or simply“cis-elements,” each of which appears to confer a different aspect ofthe overall control of gene expression (Strittmatter et al., Proc. Nat.Acad. Sci. USA 84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987;Benfey et al., EMBO J. 9:1677-1684, 1990).“cis elements” bindtrans-acting protein factors that regulate transcription. Some ciselements bind more than one factor, and trans-acting transcriptionfactors may interact with different affinities with more than one ciselement (Johnson and McKnight, Ann. Rev. Biochem. 58:799-839, 1989).Plant transcription factors, corresponding cis elements, and analysis oftheir interaction are discussed, for example, in: Martin, Curr. OpinionsBiotech. 7:130-138, 1996; Murai, In: Methods in Plant Biochemistry andMolecular Biology, Dashek, ed., CRC Press, 1997, pp. 397-422; andMethods in Plant Molecular Biology, Maliga et al., eds., Cold SpringHarbor Press, 1995, pp. 233-300. The promoter sequences of the presentinvention can contain “cis elements” that can confer or modulate geneexpression.

[0064] Cis elements can be identified by a number of techniques,including deletion analysis, i.e., deleting one or more nucleotides fromthe 5′ end or internal to a promoter; DNA binding protein analysis usingDNase I footprinting, methylation interference, electrophoresismobility-shift assays, in vivo genomic footprinting by ligation-mediatedPCR, and other conventional assays; or by sequence similarity with knowncis element motifs by conventional sequence comparison methods. The finestructure of a cis element can be further studies by mutagenesis (orsubstitution) of one or more nucleotides or by other conventionalmethods. See, e.g., Methods in Plant Biochemistry and Molecular Biology,Dashek, ed., CRC Press, 1997, pp. 397-422; and Methods in PlantMolecular Biology, Maliga et al., eds., Cold Spring Harbor Press, 1995,pp. 233-300.

[0065] Cis elements can be obtained by chemical synthesis or by cloningfrom promoters that includes such elements, and they can be synthesizedwith additional flanking sequences that contain useful restrictionenzyme sites to facilitate subsequence manipulation. In one embodiment,the promoters are comprised of multiple distinct “cis-actingtranscriptional regulatory elements,” or simply “cis-elements,” each ofwhich appears to confer a different aspect of the overall control ofgene expression (Strittmatter et al., Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Ellis et al., EMBO J. 6:11-16, 1987; Benfey et al.,EMBO J. 9:1677-1684, 1990). In a preferred embodiment sequence regionscomprising “cis elements” of the nucleic acid sequences of SEQ ID NO: 3are identified using computer programs designed specifically to identifycis elements, or domains or motifs within sequences.

[0066] The present invention includes cis elements of SEQ ID NO: 3 orhomologues of cis elements known to effect gene regulation that showhomology with the nucleic acid sequences of the present invention. Anumber of such elements are known in the literature, such as elementsthat are regulated by numerous factors such as light, heat, or stress;elements that are regulated or induced by pathogens or chemicals, andthe like. Such elements may either positively or negatively regulatedgene expression, depending on the conditions. Examples of cis elementswould include but are not limited to oxygen responsive elements (Cowenet al., J. Biol. Chem. 268(36):26904, 1993), light regulatory elements(see for example, Bruce and Quaill, Plant Cell 2(11): 1081. 1990, andBruce et al., EMBO J. 10:3015, 1991, a cis element reponsive to methyljasmonate treatment (Beaudoin and Rothstein, Plant Mol. Biol. 33:835,1997, salicylic acid responsive elements (Strange et al., Plant J.11:1315, 1997, heat shock response elements (Pelham et al., TrendsGenet. 1:31, 1985, elements responsive to wounding and abiotic stress(Loace et al., Proc. Natl. Acad. Sci. U. S. A. 89:9230, 1992; Mhiri etal., Plant Mol. Biol. 33:257, 1997), low temperature elements (Baker etal., Plant Mol. Biol. 24:701, 1994; Jiang et al., Plant Mol. Biol.30:679, 1996; Nordin et al., Plant Mol. Biol. 21:641, 1993; Zhou et al.,J. Biol. Chem. 267:23515, 1992), and drought responsive elements,(Yamaguchi et al., Plant Cell 6:251-264, 1994; Wang et al., Plant Mol.Biol. 28:605, 1995; Bray E. A. Trends in Plant Science 2:48, 1997).

[0067] The present invention therefore encompasses “cis elements” or“motifs” of the disclosed polynucleotide sequence and the region of thedisclosed sequence that comprises the motifs. The polynucleotide regionsof the present invention are less than the full length of the sequenceencompassed by SEQ ID NO: 3, and can contain one or more regulatoryelements including but not limited to cis elements or motifs that arecapable of enhancing transcription of operably linked DNA sequences inplant root cells and tissues.

[0068] Plant promoters can include promoters produced through themanipulation of known promoters to produce synthetic, chimeric, orhybrid promoters. Such promoters can also combine cis elements from oneor more promoters, for example, by adding a heterologous regulatorysequence to an active promoter with its own partial or completeregulatory sequences (Ellis et al., EMBO J. 6:11-16, 1987; Strittmatterand Chua, Proc. Nat. Acad. Sci. USA 84:8986-8990, 1987; Poulsen andChua, Mol. Gen. Genet. 214:16-23, 1988; Comai et al., Plant. Mol. Biol.15:373-381, 1991). Chimeric promoters have also been developed by addinga heterologous regulatory sequence to the 5′ upstream region of aninactive, truncated promoter, i.e., a promoter that includes only thecore TATA and, optionally, the CCAAT elements (Fluhr et al., Science232:1106-1112, 1986; Strittmatter and Chua, Proc. Nat. Acad. Sci. USA84:8986-8990, 1987; Aryan et al., Mol. Gen. Genet. 225:65-71, 1991). Thedesign, construction, and use of chimeric or hybrid promoters comprisingat least one cis element of SEQ ID NO: 3 for modulating the expressionof operably linked nucleic acid sequences is also encompassed by thepresent invention.

[0069] The promoter sequences, fragments, regions or cis elementsthereof of SEQ ID NO: 3 are capable of transcribing operably linked DNAsequences in multiple tissues and can selectively regulate expression ofgenes in these tissues. For a number of agronomic traits, expression ofa gene or genes of interest is desirable in multiple tissues in order toconfer the desired characteristic(s). The availability of suitablepromoters that regulate transcription of operably linked genes inselected target tissues of interest is important since it may not bedesirable to have expression in every tissue, but only in certaintissues. For example, if one desires to control an insect pest thattargets particular tissues, it would be of interest to express thedesired gene product(s) in those tissues. For herbicide tolerance, itmay be desirable to have a promoter that transcribes operably linkedgenes in a manner that confers herbicide tolerance at the desired levelsin both vegetative and reproductive tissues. Consequently, it isimportant to have a wide variety of choices of 5′ regulatory elementsfor any plant biotechnology strategy. Herbicides that are incorporatedinto the soil and function as preemergence herbicides function byinhibition the emerging root or hypocotyl. A root enhanced promoterlinked to a preemergence resistance gene that functions in the emergingroot is useful in a method to enhance crop tolerance to thesepreemergence herbicides.

[0070] The advent of genomics, which comprises molecular andbioinformatics techniques, has resulted in rapid sequencing and analysesof a large number of DNA samples from a vast number of targets,including but not limited to plant species of agronomic importance. Toidentify the nucleic acid sequences of the present invention from adatabase or collection of cDNA sequences, the first step involvesconstructing cDNA libraries from specific plant tissue targets ofinterest. Briefly, the cDNA libraries are first constructed from thesetissues that are harvested at a particular developmental stage, or underparticular environmental conditions. By identifying differentiallyexpressed genes in plant tissues at different developmental stages, orunder different conditions, the corresponding regulatory sequences ofthose genes can be identified and isolated. Transcript imaging enablesthe identification of tissue-preferred sequences based on specificimaging of nucleic acid sequences from a cDNA library. By transcriptimaging as used herein is meant an analysis that compares the abundanceof expressed genes in one or more libraries. The clones contained withina cDNA library are sequenced and the sequences compared with sequencesfrom publicly available databases. Computer-based methods allows theresearcher to provide queries that compare sequences from multiplelibraries. The process enables quick identification of clones ofinterest compared with conventional hybridization subtraction methodsknown to those of skill in the art.

[0071] Using conventional methodologies, cDNA libraries can beconstructed from the mRNA of a given tissue or organism using poly dTprimers and reverse transcriptase (Efstratiadis, et al., Cell 7:279,1976; Higuchi, et al., Proc. Natl. Acad. Sci. (U.S.A.) 73:3146, 1976;Maniatis, et al., Cell 8:163, 1976; Land et al., Nucleic Acids Res.9:2251, 1981; Okayama, et al., Mol. Cell. Biol. 2:161, 1982; Gubler, etal., Gene 25:263, 1983).

[0072] Several methods can be employed to obtain full-length cDNAconstructs. For example, terminal transferase can be used to addhomopolymeric tails of dC residues to the free 3′ hydroxyl groups (Land,et al., Nucleic Acids Res. 9:2251, 1981). This tail can then behybridized by a poly dG oligo that can act as a primer for the synthesisof full length second strand cDNA. Okayama and Berg, report a method forobtaining full length cDNA constructs. This method has been simplifiedby using synthetic primer-adapters that have both homopolymeric tailsfor priming the synthesis of the first and second strands andrestriction sites for cloning into plasmids (Coleclough, et al., Gene34:305, 1985) and bacteriophage vectors (Krawinkel, et al., NucleicAcids Res. 14:1913, 1986; and Han, et al., Nucleic Acids Res. 15:6304,1987).

[0073] These strategies can be coupled with additional strategies forisolating rare mRNA populations. For example, a typical mammalian cellcontains between 10,000 and 30,000 different mRNA sequences. Davidson,Gene Activity in Early Development, 2nd ed., Academic Press, New York,1976. The number of clones required to achieve a given probability thata low-abundance mRNA will be present in a cDNA library isN=(1n(1−P))/(1n(1−1/n)) where N is the number of clones required, P isthe probability desired, and 1/n is the fractional proportion of thetotal mRNA that is represented by a single rare mRNA (Sambrook, etal.,1989).

[0074] One method to enrich preparations of mRNA for sequences ofinterest is to fractionate by size. One such method is to fractionate byelectrophoresis through an agarose gel (Pennica, et al., Nature 301:214,1983). Another such method employs sucrose gradient centrifugation inthe presence of an agent, such as methylmercuric hydroxide, thatdenatures secondary structure in RNA (Schweinfest, et al., Proc. Natl.Acad. Sci. (U.S.A.) 79:4997-5000, 1982).

[0075] A frequently adopted method is to construct equalized ornormalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705, 1990;Patanjali, S. R. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1943, 1991).Typically, the cDNA population is normalized by subtractivehybridization. Schmid, et al., J. Neurochem. 48:307, 1987; Fargnoli, etal., Anal. Biochem. 187:364, 1990; Travis, et al., Proc. Natl. Acad. Sci(U.S.A.) 85:1696, 1988; Kato, Eur. J. Neurosci. 2:704, 1990; andSchweinfest, et al., Genet. Anal. Tech. Appl. 7:64, 1990). Subtractionrepresents another method for reducing the population of certainsequences in the cDNA library (Swaroop, et al., Nucleic Acids Res.19:1954, 1991). Normalized libraries can be constructed using the Soaresprocedure (Soares et al., Proc. Natl. Acad. Sci. (U. S. A.) 91:9228,1994). This approach is designed to reduce the initial 10,000-foldvariation in individual cDNA frequencies to achieve abundances withinone order of magnitude while maintaining the overall sequence complexityof the library. In the normalization process, the prevalence ofhigh-abundance cDNA clones decreases dramatically, clones with mid-levelabundance are relatively unaffected, and clones for rare transcripts areeffectively increased in abundance.

[0076] ESTs can be sequenced by a number of methods. Two basic methodscan be used for DNA sequencing, the chain termination method of Sangeret al., Proc. Natl. Acad. Sci. (U.S.A.) 74: 5463, 1977 and the chemicaldegradation method of Maxam and Gilbert, Proc. Nat. Acad. Sci. (U.S.A.)74: 560, 1977. Automation and advances in technology such as thereplacement of radioisotopes with fluorescence-based sequencing havereduced the effort required to sequence DNA (Craxton, Methods, 2: 20,1991; Ju et al., Proc. Natl. Acad. Sci. (U.S.A.) 92: 4347, 1995; Taborand Richardson, Proc. Natl. Acad. Sci. (U.S.A.) 92: 6339, 1995).Automated sequencers are available from a number of manufacturers, forexample, Pharmacia Biotech, Inc., Piscataway, N.J. (Pharmacia ALF),LI-COR, Inc., Lincoln, Nebr. (LI-COR 4,000) and Millipore, Bedford,Mass. (Millipore BaseStation).

[0077] ESTs longer than 150 bp have been found to be useful forsimilarity searches and mapping. (Adams, et al., Science 252:1651, 1991.EST sequences normally range from 150-450 bases. This is the length ofsequence information that is routinely and reliably generated usingsingle run sequence data. Typically, only single run sequence data isobtained from the cDNA library, Adams, et al., Science 252:1651, 1991.Automated single run sequencing typically results in an approximately2-3% error or base ambiguity rate. (Boguski, et al., Nature Genetics,4:332, 1993).

[0078] EST databases have been constructed or partially constructedfrom, for example, C. elegans (McCombrie, et al., Nature Genetics 1:124, 1992, human liver cell line HepG2 (Okubo, et al., Nature Genetics2:173, 1992); human brain RNA (Adams, et al., Science 252:1651, 1991;Adams, et al., Nature 355:632, 1992); Arabidopsis, (Newman, et al.,Plant Physiol. 106:1241, 1994); and rice (Kurata, et al., NatureGenetics 8:365, 1994). The present invention uses ESTs from a number oflibraries prepared from corn root and leaf tissues as a tool for theidentification of genes expressed in these tissues, which thenfacilitates the isolation of 5′ regulatory sequences such as promotersthat regulate the genes.

[0079] Computer-based sequence analyses can be used to identifydifferentially expressed sequences including but not limited to thosesequences expressed in one tissue compared with another tissue. Forexample, a different set of sequences can be found from cDNA isolatedfrom plant tissue isolated from root tissue versus leaf tissue.Accordingly, sequences can be compared from cDNA libraries prepared fromplants grown under different environmental or physiological conditions.Once the preferred sequences are identified from the cDNA library ofinterest, the genomic clones can be isolated from a genomic libraryprepared from the plant tissue, and corresponding regulatory sequencesincluding but not limited to 5′ regulatory sequences can be identifiedand isolated.

[0080] In one preferred embodiment, expressed sequence tags (EST)sequences from a variety of cDNA libraries are catalogued in a sequencedatabase. This database is used to identify promoter targets from aparticular tissue of interest. The selection of expressed sequence tagsfor subsequent promoter isolation is reflective of the presence of oneor more sequences among the representative ESTs from a random samplingof an individual cDNA library, or a collection of cDNA libraries. Forexample, the identification of regulatory sequences that regulate theexpression of transcripts in leaf and root tissue is conducted byidentifying ESTs found in leaf and root cDNA libraries and absent or inlower abundance in other cDNA libraries and the expression profile for agiven EST is assessed. By abundance as used herein is meant the numberof times a clone or cluster of clones appears in a library. Thesequences that are enhanced or in high abundance in a specific tissue ororgan that represent a target expression profile are identified in thismanner and primers can be designed from the identified EST sequences. APCR-based approach can be used to amplify flanking regions from agenomic library of the target plant of interest. A number of methods areknown to those of skill in the art to amplify unknown DNA sequencesadjacent to a core region of known sequence. Methods include but are notlimited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR and genomewalking approaches.

[0081] In a preferred embodiment, genomic DNA ligated to an adapter issubjected to a primary round of PCR amplification with a gene-specificprimer and a primer that anneals to the adapter sequence. The PCRproduct is next used as the template for a nested round of PCRamplification with a second gene-specific primer and second adapter. Theresulting fragments from the nested PCR reaction are then isolated,purified and subcloned into an appropriate vector. The fragments aresequenced and the translational start sites can be identified when theEST is derived from a truncated cDNA. The fragments can be cloned intoplant expression vectors as transcriptional or translational fusionswith a reporter gene such as β-glucuronidase (GUS). The constructs canbe tested in transient analyses and subsequently the 5′ regulatoryregions are operably linked to other genes and regulatory sequences ofinterest in a suitable plant transformation vector and the transformedplants are analyzed for the expression of the gene(s) of interest by anynumber of methods known to those of skill in the art.

[0082] Any plant can be selected for the identification of genes andregulatory sequences. Examples of suitable plant targets for theisolation of genes and regulatory sequences would include but are notlimited to Acadia, alfalfa, apple, apricot, Arabidopsis, artichoke,arugula, asparagus, avocado, banana, barley, beans, beet, blackberry,blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,carrot, cassava, castorbean, cauliflower, celery, cherry, chicory,cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton,cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel,figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit,lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon,mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive,onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radiscchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams, and zucchini. Particularlypreferred plant targets would include corn, cotton, soybean, and wheat.

[0083] The nucleic acid molecules of the present invention are isolatedfrom corn (Zea mays). The corn plant develops about 20-21 leaves, silksabout 65 days post-emergence, and matures about 125 days post-emergence.Normal corn plants follow a general pattern of development, but the timeinterval between different stages and morphology varies betweendifferent hybrids, growth and environmental conditions.

[0084] There are a number of identifiable stages in corn plantdevelopment. The stages are defined as vegetative (V) and reproductive(R) stages. Subdivisions of the V stages are numerically designated asV1, V2,V3, etc., through V(n) where (n) represents the last leaf stagebefore tasseling (VT) and the first V stage is the emergence (VE) stage.For example, VE is the emergence from the soil of a seedling leaf, V1represents the first true leaf, V2 represents the second leaf, etc. Thereproductive stages include the first appearance of silk to the matureseed and are represented as follows: R1 is silking, R2 is blistering, R3is the milk stage, R4 is the dough stage, R5 is the dent stage, and R6is physiological maturity (see for example, Ritchie S W et al. (1986)How a Corn Plant Develops, Iowa State University of Science andTechnology Cooperative Exension Service, Ames, Iowa 48: 1-21).

[0085] Any method that allows a differential comparison betweendifferent types or classes of sequences can be used to isolate genes orregulatory sequences of interest. For example in one differentialscreening approach, a cDNA library from mRNA isolated from a particulartissue can be prepared in a bacteriophage host using a commerciallyavailable cloning kit. The plaques are spread onto plates containing alawn of a bacterial host such as E. coli to generate bacteriophageplaques. About 10⁵-10⁶ plaques can be lifted onto DNA binding membranes.Duplicate membranes are probed using probes generated from mRNA from thetarget and non-target or background tissue. The probes are labeled tofacilitate detection after hybridization and development. Plaques thathybridize to target tissue-derived probes but not to non-target tissuederived probes that display a desired differential pattern of expressioncan be selected for further analysis. Genomic DNA libraries can also beprepared from a chosen species by partial digestion with a restrictionenzyme and size selecting the DNA fragments within a particular sizerange. The genomic DNA can be cloned into a suitable vector includingbut not limited to a bacteriophage, and prepared using a suitable kit asdescribed earlier (see for example, Stratagene, La Jolla, Calif. orGibco BRL, Gaithersburg, Md.).

[0086] Differential hybridization techniques as described are well knownto those of skill in the art and can be used to isolate a desired classof sequences. By classes of sequences as used herein is meant sequencesthat can be grouped based on a common identifier including but notlimited to sequences isolated from a common target plant, a commonlibrary, or a common plant tissue type. In a preferred embodiment,sequences of interest are identified based on sequence analyses andquerying of a collection of diverse cDNA sequences from libraries ofdifferent tissue types.

[0087] A number of methods used to assess gene expression are based onmeasuring the mRNA level in an organ, tissue, or cell sample. Typicalmethods include but are not limited to RNA blots, ribonucleaseprotection assays and RT-PCR. In another preferred embodiment, ahigh-throughput method is used whereby regulatory sequences areidentified from a transcript profiling approach. The development of cDNAmicroarray technology enables the systematic monitoring of geneexpression profiles for thousands of genes (Schena et al, Science, 270:467, 1995). This DNA chip-based technology arrays thousands of cDNAsequences on a support surface. These arrays are simultaneouslyhybridized to a-multiple of labeled cDNA probes prepared from RNAsamples of different cell or tissue types, allowing direct comparativeanalysis of expression. This technology is first demonstrated byanalyzing 48 Arabidopsis genes for differential expression in roots andshoots (Schena et al, Science, 270:467, 1995). More recently, theexpression profiles of over 1400 genes are monitored using cDNAmicroarrays (Ruan et al, The Plant Journal 15:821, 1998). Microarraysprovide a high-throughput, quantitative and reproducible method toanalyze gene expression and characterize gene function. The transcriptprofiling approach using microarrays thus provides another valuable toolfor the isolation of regulatory sequences such as promoters associatedwith those genes.

[0088] The present invention uses high throughput sequence analyses toform the foundation of rapid computer-based identification of sequencesof interest. Those of skill in the art are aware of the resourcesavailable for sequence analyses. Sequence comparisons can be undertakenby determining the similarity of the test or query sequence withsequences in publicly available or proprietary databases (“similarityanalysis”) or by searching for certain motifs (“intrinsic sequenceanalysis”) (e.g. cis elements) (Coulson, Trends in Biotechnology, 12:76,1994; Birren, et al., Genome Analysis, 1:543, 1997).

[0089] The nucleotide sequences provided in SEQ ID NO: 3 or fragmentsthereof, or complements thereof, or a nucleotide sequence at least 90%substantial identity, preferably 95% substantial identity even morepreferably 99% or 100% substantial identity to the sequence provided inSEQ ID NO: 3 or fragment thereof, or cis element thereof, or complementof the sequence thereof, can be “provided” in a variety of mediums tofacilitate use. Such a medium can also provide a subset thereof in aform that allows one of skill in the art to examine the sequences.

[0090] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, “computer readable media” refers to any medium that can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard disc,storage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. One of skill in theart can readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention.

[0091] By providing one or more of nucleotide sequences of the presentinvention, those of skill in the art can routinely access the sequenceinformation for a variety of purposes. Computer software is publiclyavailable that allows one of skill in the art to access sequenceinformation provided in a computer readable medium. Examples of publicdatabases would include but is not limited to the DNA Database of Japan(DDBJ) (http://www.ddbj.nig.ac.jp/);Genbank(http://www.ncbi.nlm.nih.gov/web/Genbank/Index.html); and the EuropeanMolecular Biology Laboratory Nucleic Acid Sequence Database(EMBL)(http://www.ebi.ac.uk/ebi_docs/embl_db.html) or versions thereof.A number of different search algorithms have been developed, includingbut not limited to the suite of programs referred to as BLAST programs.There are five implementations of BLAST, three designed for nucleotidesequence queries (BLASTN, BLASTX, and TBLASTX) and two designed forprotein sequence queries (BLASTP and TBLASTN) (Coulson, Trends inBiotechnology, 12:76-80, 1994; Birren, et al., Genome Analysis, 1:543,1997).

[0092] Any program designed for motif searching also has utility in thepresent invention. Sequence analysis programs designed for motifsearching can be used for identification of cis elements. Preferredcomputer programs would include but are not limited to MEME, SIGNALSCAN, and GENESCAN. Meme is a program that identifies conserved motifs(either nucleic acid or peptide) in a group of unaligned sequences. Memesaves these motifs as a set of profiles. These profiles can be used tosearch a database of sequences. A MEME algorithm (version 2.2) can befound in version 10.0 of the GCG package; MEME (T. Bailey and C. Elkan,Machine Learning, 21(1-2):51-80,1995 and the location of the website isas follows: (http:H/www.sdsc.edu/MEME/meme/website/COPYRIGHT.html.).SignalScan is a program that identifies known motifs in the testsequences using information from other motif databases (Prestridge, D.S., CABIOS 7, 203-206 (1991). SignalScan version 4.0 information isavailable at the following website:http://biosci.cbs.umn.edu/software/sigscan.html. The ftp site for SignalScan is ftp://biosci.cbs.umn.edu/software/sigscan.html. Databases usedwith Signal Scan include PLACE (http://www.dna.affrc.go.ip/htdocs/PLACE(Higo et al., Nucleic Acids Research 27(1):297-300 (1999) and TRANSFAC(Heinemeye, X. et al., Nucleic Acid Research 27(1):318-322) that can befound at the following website: http://transfac.gbf.de/. GeneScan isanother suitable program for motif searching (Burge, C and Karlin, S. J.Mol. Biol. 268, 78-94 (1997) and version 1.0 information is available atthe following website: http://gnomic.stanford.edu/GENESCANW.html. Asused herein, “a target structural motif”, or “target motif” refers anyrationally selected sequence or combination of sequences in which thesequence(s) are chosen based on a three-dimensional configuration thatis formed upon the folding of the target motif. There are a variety oftarget motifs known to those of skill in the art. Protein target motifsinclude but are not limited to, enzymatic active sites and signalsequences. Preferred target motifs of the present invention wouldinclude but are not limited to promoter sequences, cis elements, hairpinstructures and other expression elements such as protein bindingsequences.

[0093] As used herein, “search means” refers to one or more programsthat are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of the sequences of the present invention thatmatch a particular target sequence or target motif. Also, multiplesequences can be compared in order to identify common regions or motifsthat may be responsible for specific functions. For example, ciselements or sequence domains that confer a specific expression profilecan be identified when multiple promoter regions of similar classes ofpromoters are aligned and analyzed by certain software packages.

[0094] The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. As used herein, a “computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. Those of skill in the art can appreciate thatany one of the available computer-based systems are suitable for use inthe present invention.

[0095] DNA molecules for use as PCR primers are designed from the cDNAsequences identified from the computer-based sequence comparisons. Thesesequences are used to extend the nucleic acid sequence using polymerasechain reaction (PCR) amplification techniques (see for example, Mulliset al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1986; Erlich, etal., European Patent Appln. 50,424; European Patent Appln. 84,796,European Patent Appln. 258,017, European Patent Appln. 237,362; Mullis,European Patent Appln. 201,184; Mullis, et al., U.S. Pat. No. 4,683,202;Erlich, U.S. Pat. No. 4,582,788; and Saiki, et al., U.S. Pat. No.4,683,194). A number of PCR amplification methods are known to those ofskill in the art, and are used to identify nucleic acid sequencesadjacent to a known sequence. For example, inverse PCR (IPCR) methods toamplify unknown DNA sequences adjacent to a core region of knownsequence have been described. Other methods are also available such ascapture PCR (Lagerstrom M., et al., PCR Methods Applic. 1: 111, 1991,and walking PCR (Parker, J D et al., Nucl. Acids Res 19:3055, 1991). Anumber of manufacturers have also developed kits based on modificationsof these methods for the purposes of identifying sequences of interest.Technical advances including improvements in primer and adapter design,improvements in the polymerase enzyme, and thermocycler capabilitieshave facilitated quicker, efficient methods for isolating sequences ofinterest.

[0096] In a preferred embodiment, the flanking sequences containing the5′ regulatory elements of the present invention are isolated using agenome-walking approach (Universal GenomeWalker™ Kit, CLONTECHLaboratories, Inc., Palo, Alto, Calif.). In brief, the purified genomicDNA is subjected to a restriction enzyme digest that produces genomicDNA fragments with ends that are ligated with GenomeWalker™ adapters.GenomeWalker™ primers are used along with gene specific primers in twoconsecutive PCR reactions (primary and nested PCR reactions) to producePCR products containing the 5′ regulatory sequences that aresubsequently cloned and sequenced.

[0097] In addition to their use in modulating gene expression, thepromoter sequences of the present invention also have utility as probesor primers in nucleic acid hybridization experiments. The nucleic-acidprobes and primers of the present invention can hybridize understringent conditions to a target DNA sequence. The term “stringenthybridization conditions” is defined as conditions under which a probeor primer hybridizes specifically with a target sequence(s) and not withnon-target sequences, as can be determined empirically. The term“stringent conditions” is functionally defined with regard to thehybridization of a nucleic-acid probe to a target nucleic acid (i.e., toa particular nucleic-acid sequence of interest) by the specifichybridization procedure discussed in Sambrook et al., 1989, at9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58;Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J.Mol. Biol. 31:349-370, 1968. Appropriate stringency conditions thatpromote DNA hybridization are, for example, 6.0×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 1989,6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged.

[0098] For example, hybridization using DNA or RNA probes or primers canbe performed at 65° C. in 6×SSC, 0.5% SDS, 5×Denhardt's, 100 μg/mLnonspecific DNA (e.g., sonicated salmon sperm DNA) with washing at0.5×SSC, 0.5% SDS at 65° C., for high stringency.

[0099] It is contemplated that lower stringency hybridization conditionssuch as lower hybridization and/or washing temperatures can be used toidentify related sequences having a lower degree of sequence similarityif specificity of binding of the probe or primer to target sequence(s)is preserved. Accordingly, the nucleotide sequences of the presentinvention can be used for their ability to selectively form duplexmolecules with complementary stretches of DNA fragments. Detection ofDNA segments via hybridization is well-known to those of skill in theart, and thus depending on the application envisioned, one will desireto employ varying hybridization conditions to achieve varying degrees ofselectivity of probe towards target sequence and the method of choicewill depend on the desired results.

[0100] The nucleic acid sequences in SEQ ID NO: 3 and any variantsthereof, are capable of hybridizing to other nucleic acid sequencesunder appropriately selected conditions of stringency. As used herein,two nucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complete complementarity. As used herein, molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the molecules is complementary to a nucleotide of the other. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low stringency”conditions. Similarly, the molecules are said to be “complementary” isthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional “highstringency” conditions. Conventional stringency conditions are describedby Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and by Haymeset al., Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C., 1985.

[0101] In a preferred embodiment, the nucleic acid sequences, SEQ ID NO:3, or a fragment, region, cis element, or oligomer of any of thesesequences, may be used in hybridization assays of other plant tissues toidentify closely related or homologous genes and associated regulatorysequences. These include but are not limited to Southern hybridizationassays on any substrate including but not limited to an appropriatelyprepared plant tissue, cellulose, nylon, or combination filter, chip, orglass slide. Such methodologies are well known in the art and areavailable in a kit or preparation that can be supplied by commercialvendors.

[0102] Of course, fragments can also be obtained by other techniquessuch as by directly synthesizing the fragment by chemical means, as iscommonly practiced by using an automated oligonucleotide synthesizer.Also, fragments can be obtained by application of nucleic acidreproduction technology, such as the PCR™ (polymerase chain reaction)technology by recombinant DNA techniques generally known to those ofskill in the art of molecular biology. Regarding the amplification of atarget nucleic-acid sequence (e.g., by PCR) using a particularamplification primer pair, “stringent PCR conditions” refer toconditions that permit the primer pair to hybridize only to the targetnucleic-acid sequence to which a primer having the correspondingwild-type sequence (or its complement) would bind and preferably toproduce a unique amplification product.

[0103] A fragment comprises at least a minimum length of identicalpolynucleotide sequence. The fragment can be used in hybridization orPCR under stringent hybridization conditions as defined above to isolatelike molecules. For example, for the present invention a fragment lengthof polynucleotide sequence is one that would have polynucleotidesequence identical to at least 36 nucleotides of SEQ ID NO: 3.

[0104] The nucleic acid sequences of the present invention can also beused as probes and primers. Nucleic acid probes and primers can beprepared based on a native gene sequence. A “probe” is an isolatednucleic acid to which is attached a conventional detectable label orreporter molecule, e.g., a radioactive isotope, ligand, chemiluminescentagent, or enzyme. “Primers” are isolated nucleic acids that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs can be used for amplification of a nucleic acidsequence, e.g., by the polymerase chain reaction (PCR) or otherconventional nucleic-acid amplification methods.

[0105] Probes and primers are generally 15 nucleotides or more inlength, preferably 20 nucleotides or more, more preferably 25nucleotides, and most preferably 30 nucleotides or more. Such probes andprimers hybridize specifically to a target DNA or RNA sequence underhigh stringency hybridization conditions and hybridize specifically to atarget native sequence of another species under lower stringencyconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the native sequence,although probes differing from the native sequence and that retain theability to hybridize to target native sequences may be designed byconventional methods. Methods for preparing and using probes and primersare described, for example, in Molecular Cloning: A Laboratory Manual,2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al.,1989”); Current Protocols in Molecular Biology, ed. Ausubel et al.,Greene Publishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (herein referred to as, “Ausubel et al., 1992); and Innis etal., PCR Protocols: A Guide to Methods and Applications, Academic Press:San Diego, 1990, herein incorporated by reference in their entirety.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). Primers and probes based on the native promotersequences disclosed herein can be used to confirm and, if necessary, tomodify the disclosed sequences by conventional methods, e.g., byre-cloning and re-sequencing.

[0106] In another embodiment, the nucleotide sequence of the promoterdisclosed herein can be modified. Those skilled in the art can createDNA molecules that have variations in the nucleotide sequence. Thenucleotide sequence of the present invention as shown in SEQ ID NO: 3may be modified or altered to enhance their control characteristics. Onepreferred method of alteration of a nucleic acid sequence is to use PCRto modify selected nucleotides or regions of sequences. These methodsare known to those of skill in the art. Sequences can be modified, forexample by insertion, deletion or replacement of template sequences in aPCR-based DNA modification approach. “Variant” DNA molecules are DNAmolecules containing changes in which one or more nucleotides of anative sequence is deleted, added, and/or substituted, preferably whilesubstantially maintaining promoter function. In the case of a promoterfragment, “variant” DNA can include changes affecting the transcriptionof a minimal promoter to which it is operably linked. Variant DNAmolecules can be produced, for example, by standard DNA mutagenesistechniques or by chemically synthesizing the variant DNA molecule or aportion thereof.

[0107] In another embodiment, the nucleotide sequences as shown in SEQID NO: 3 includes any length of said nucleotide sequences that iscapable of regulating an operably linked DNA sequence. For example, thesequences as disclosed in SEQ ID NO: 3 may be truncated or portionsdeleted and still be capable of regulating transcription of an operablylinked DNA sequence. In a related embodiment, a cis element of thedisclosed sequences may confer a particular specificity such asconferring enhanced expression of operably linked DNA sequences incertain tissues and therefore is also capable of regulatingtranscription of operably linked DNA sequences. Consequently, anysequence fragments, portions, or regions of the disclosed sequences ofSEQ ID NO: 3 can be used as regulatory sequences, including but notlimited to cis elements or motifs of the disclosed sequences. Forexample, one or more base pairs may be deleted from the 5′ or 3′ end ofa promoter sequence to produce a “truncated” promoter. One or more basepairs can also be inserted, deleted, or substituted internally to apromoter sequence. Promoters can be constructed such that promoterfragments or elements are operably linked, for example, by placing sucha fragment upstream of a minimal promoter. A minimal or basal promoteris a piece of DNA that is capable of recruiting and binding the basaltranscription machinery. One example of basal transcription machinery ineukaryotic cells is the RNA polymerase II complex and its accessoryproteins. The enzymatic components of the basal transcription machineryare capable of initiating and elongating transcription of a given gene,utilizing a minimal or basal promoter. That is, there are not addedcis-acting sequences in the promoter region that are capable ofrecruiting and binding transcription factors that modulatetranscription, e.g., enhance, repress, render transcriptionhormone-dependent, etc. Substitutions, deletions, insertions or anycombination thereof can be combined to produce a final construct.

[0108] Native or synthetic nucleic acids according to the presentinvention can be incorporated into recombinant nucleic acid constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. In one preferred embodiment, the nucleotide sequences ofthe present invention as shown in SEQ ID NO: 3 or fragments, variants,or derivatives thereof are incorporated into an expression vectorcassette that includes the promoter regions of the present inventionoperably linked to a genetic component such as a selectable, screenable,or scorable marker gene. The disclosed nucleic acid sequences of thepresent invention are preferably operably linked to a genetic componentsuch as a nucleic acid that confers a desirable characteristicassociated with plant morphology, physiology, growth and development,yield, nutritional enhancement, disease or pest resistance, orenvironmental or chemical tolerance. These genetic components such asmarker genes or agronomic genes of interest can function in theidentification of a transformed plant cell or plant, or a produce aproduct of agronomic utility.

[0109] In a preferred embodiment, one genetic component produces aproduct that serves as a selection device and functions in a regenerableplant tissue to produce a compound that would confer upon the planttissue resistance to an otherwise toxic compound. Genes of interest foruse as a selectable, screenable, or scorable marker gene would includebut are not limited to the coding sequence for β-glucuronidase (GUS),the coding sequence for green fluorescent protein (GFP), the codingsequence for luciferase (LUX), antibiotic, or herbicide tolerance genes.Examples of transposons and associated antibiotic resistance genesinclude the transposons Tns (bla), Tn5 (nptII), Tn7 (dhfr), penicillins,kanamycin (and neomycin, G418, bleomycin); methotrexate (andtrimethoprim); chloramphenicol; kanamycin and tetracycline.

[0110] Characteristics useful for selectable markers in plants have beenoutlined in a report on the use of microorganisms (Advisory Committee onNovel Foods and Processes, July 1994). These include stringent selectionwith minimum number of nontransformed tissues, large numbers ofindependent transformation events with no significant interference withthe regeneration, application to a large number of species, andavailability of an assay to score the tissues for presence of themarker.

[0111] A number of selectable marker genes are known in the art andseveral antibiotic resistance markers satisfy these criteria, includingthose resistant to kanamycin (nptII), hygromycin B (aph IV),streptomycin or spectinomycin (aad, spec/strep ) and gentamycin (aac3and aacC4). Useful dominant selectable marker genes include genesencoding antibiotic resistance genes, e.g., resistance to hygromycin,kanamycin, bleomycin, G418, streptomycin or spectinomycin; and herbicideresistance genes (e.g., phosphinothricin acetyltransferase, mutant ALS,class II EPSPS and modified class I EPSPS). A useful strategy forselection of transformants for herbicide resistance is described, e.g.,in Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vols. I-III,Laboratory Procedures and Their Applications Academic Press, New York,1984. Particularly preferred selectable marker genes for use in thepresent invention would genes that confer resistance to compounds suchas antibiotics like kanamycin, and herbicides like glyphosate(Della-Cioppa et al., Bio/Technology 5(6), 1987, U.S. Pat. No.5,463,175, U.S. Pat. No. 5,633,435). Other selection-devices can also beimplemented and would still fall within the scope of the presentinvention.

[0112] For the practice of the present invention, conventionalcompositions and methods for preparing and using vectors and host cellsare employed, as discussed, inter alia, in Sambrook et al., 1989). In apreferred embodiment, the host cell is a plant cell. A number of vectorssuitable for stable transfection of plant cells or for the establishmentof transgenic plants have been described in, e.g., Pouwels et al.,Cloning Vectors: A Laboratory Manual, 1985, supp. 1987); Weissbach andWeissbach, Methods for Plant Molecular Biology, Academic Press, 1989;Gelvin et al., Plant Molecular Biology Manual, Kluwer AcademicPublishers, 1990; and R. R. D. Croy, Plant Molecular Biology LabFax,BIOS Scientific Publishers, 1993. Plant expression vectors can include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences. They can also can include aselectable marker as described to allow selection of host cellscontaining the expression vector. Such plant expression vectors alsotypically contain a promoter regulatory region (e.g., a regulatoryregion controlling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and apolyadenylation signal. Other sequences, of bacterial origin are alsoincluded to allow the vector to be cloned in a bacterial host. Thevector will also typically contain a broad host range prokaryotic originof replication. In a particularly preferred embodiment, the host cell isa plant cell and the plant expression vector comprises a promoter regionas disclosed in SEQ ID NO: 3, an operably linked transcribable sequence,and a transcription termination sequence. Other regulatory sequences canalso be included such as 5′ non-translated leaders, in addition torestriction enzyme sites for cloning purposes.

[0113] Promoters have utility for plant gene expression for any gene ofinterest including but not limited to selectable markers, scorablemarkers, genes for pest tolerance, disease tolerance, herbicidetolerance, nutritional enhancements and any other gene, coding sequenceor noncoding sequence of agronomic interest. Examples of constitutivepromoters useful for plant gene expression include, but are not limitedto, the cauliflower mosaic virus (CaMV) 35S promoter, which confersconstitutive, high-level expression in most plant tissues (see, e.g.,Odel et al., Nature 313:810, 1985), including monocots (see, e.g.,Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen.Genet. 220:389, 1990); the nopaline synthase promoter (An et al., PlantPhysiol. 88:547, 1988) and the octopine synthase promoter (Fromm et al.,Plant Cell 1:977, 1989) and the figwort mosaic virus (FMV) promoter(U.S. Pat. No. 6,018,100).

[0114] A variety of plant gene promoters that are regulated in responseto environmental, hormonal, chemical, and/or developmental signals canbe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., Plant Physiol.88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al.,Plant Cell 1:471, 1989; maize RbcS promoter, Schaffner and Sheen, PlantCell 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpsonet al., EMBO J. 4:2723, 1985), (3) hormones, such as abscisic acid(Marcotte et al., Plant Cell 1:969, 1989), (4) wounding (e.g., wunI,Siebertz et al., Plant Cell 1:961, 1989); or (5) chemicals such asmethyl jasmonate, salicylic acid, or Safener. It may also beadvantageous to employ (6) organ-specific promoters (e.g., Roshal etal., EMBO J. 6:1155, 1987; Schernthaner et al., EMBO J. 7:1249, 1988;Bustos et al., Plant Cell 1:839, 1989).

[0115] The promoter of the present invention is a plant promoter that iscapable of transcribing operatively linked DNA sequences in multipleplant tissues, however, preferably expression is enhanced in root cellsand root tissues relative to other plant cells and tissues.

[0116] Plant expression vectors can include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression vectors may include additional regulatory sequences from the3′-untranslated region (3′ UTR) of plant genes (Thornburg et al., Proc.Natl. Acad. Sci. USA 84:744 (1987); An et al., Plant Cell 1:115 (1989),e.g., a 3′ UTR to increase mRNA stability of the mRNA, such as the PI-IItermination region of potato or the octopine or nopaline synthase 3′termination regions). 5′ untranslated regions (5′UTR) of a mRNA can playan important role in translation initiation and can also be a geneticcomponent in a plant expression vector. For example, non-translated 5′leader sequences derived from heat shock protein genes have beendemonstrated to enhance gene expression in plants (see, for example U.S.Pat. No. 5,362,865 herein incorporated by reference in its entirety).These additional upstream and downstream regulatory sequences may bederived from a source that is native or heterologous with respect to theother elements present on the expression vector.

[0117] The promoter sequence of the present invention is used to controlgene expression in plant cells. The disclosed promoter sequencecomprises genetic components that are part of DNA constructs used inplant transformation. The promoter sequences of the present inventioncan be used with any suitable plant transformation plasmid or vectorcontaining a selectable or screenable marker and associated regulatoryelements, as described, along with one or more nucleic acids expressedin a manner sufficient to confer a particular desirable trait. Examplesof suitable structural genes of agronomic interest envisioned by thepresent invention would include but are not limited to one or more genesfor insect tolerance, such as Bacillus thuringiensis insecticidalprotein genes, disease tolerance such as genes for fungal diseasecontrol, bacterial disease control and nematode control, herbicidetolerance e.g., genes conferring glyphosate tolerance, phosphinothricinetolerance, ALS inhibitor tolerance, atrazine tolerance, acteoclortolerance, alaclor tolerance, metoalaclor tolerance, isoxaflutoletolerance, and genes for quality improvements such as yield, nutritionalenhancements, environmental or stress tolerances (e.g. drought,nutrient, heat, cold, pollution) or any desirable changes in plantphysiology, growth, development, morphology or plant product(s).

[0118] Alternatively, the DNA coding sequences can effect thesephenotypes by encoding a non-translatable RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev. 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillitoe, Mol.Biotech. 7:125, 1997). Thus, any gene that produces a protein or mRNAthat expresses a phenotype or morphology change of interest are usefulfor the practice of the present invention.

[0119] In addition to regulatory elements or sequences located upstream(5′) or within a DNA sequence, there are downstream (3′) sequences thataffect gene expression and thus the term regulatory sequence as usedherein refers to any nucleotide sequence located upstream, within, ordownstream to a DNA sequence that controls, mediates, or affectsexpression of a gene product in conjunction with the protein syntheticapparatus of the cell.

[0120] The promoter sequences of the present invention may be modified,for example for expression in other plant systems. In another approach,novel hybrid promoters can be designed or engineered by a number ofmethods. Many promoters contain upstream sequences that activate,enhance or define the strength and/or specificity of the promoter(Atchison, Ann. Rev. Cell Biol. 4:127, 1988). T-DNA genes, for examplecontain “TATA” boxes defining the site of transcription initiation andother upstream elements located upstream of the transcription initiationsite modulate transcription levels (Gelvin, In Transgenic Plants (Kung,S. -D. And Us, R., eds), San Diego: Academic Press, pp.49-87, 1988).Another chimeric promoter combined a trimer of the octopine synthase(ocs) activator to the mannopine synthase (mas) activator plus promoterand reported an increase in expression of a reporter gene (Min Ni etal., The Plant Journal 7:661, 1995). The upstream regulatory sequencesof the present invention can be used for the construction of suchchimeric or hybrid promoters. Methods for construction of variantpromoters of the present invention include, but are not limited to,combining control elements of different promoters or duplicatingportions or regions of a promoter (see for example, U.S. Pat. No.4,990,607, U.S. Pat. No. 5,110,732, and U.S. Pat. No. 5,097,025, hereinincorporated by reference in their entirety). Those of skill in the artare familiar with the standard resource materials that describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolation ofgenes, (see for example Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 1989; Mailga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press, 1995; Birren et al., GenomeAnalysis: volume 1, Analyzing DNA, (1997); volume 2, Detecting Genes,(1998); volume 3, Cloning Systems, (1999), volume 4, Mapping Genomes,(1999), Cold Spring Harbor, N.Y.).

[0121] The promoter sequences of the present invention can beincorporated into an expression vector using screenable or scorablemarkers as described and tested in transient analyses that provide anindication of gene expression in stable plant systems. Methods oftesting gene expression in transient assays are known to those of skillin the art. Transient expression of marker genes has been reported usinga variety of plants, tissues and DNA delivery systems. For example,types of transient analyses can include but are not limited to directgene delivery via electroporation or particle bombardment of tissues inany transient plant assay using any plant species of interest. Suchtransient systems would include but are not limited to protoplasts fromsuspension cultures in wheat (Zhou et al., Plant Cell Reports 12:612,1993), electroporation of leaf protoplasts of wheat (Sethi et al., J.Crop Sci. 52: 152, 1983); electroporation of protoplast prepared fromcorn tissue (Sheen, J., Plant Cell 3: 225, 1991), or particlebombardment of specific tissues of interest. The present inventionencompasses the use of any transient expression system to evaluateregulatory sequences operatively linked to selected reporter genes,marker genes or agronomic genes of interest. Examples of plant tissuesenvisioned to test in transients via an appropriate delivery systemwould include but are not limited to leaf base tissues, callus,cotyledons, roots, endosperm, embryos, floral tissue, pollen, andepidermal tissue.

[0122] Any scorable or screenable marker can be used in a transientassay. Preferred marker genes for transient analyses of the promoters or5′ regulatory sequences of the present invention include a GUS gene(coding sequence for β-glucuronidase) or a GFP gene (coding sequence forgreen fluorescent protein). The expression vectors containing the 5′regulatory sequences operably linked to a marker gene are delivered tothe tissues and the tissues are analyzed by the appropriate mechanism,depending on the marker. The quantitative or qualitative analyses areused as a tool to evaluate the potential expression profile of the 5′regulatory sequences when operatively linked to genes of agronomicinterest in stable plants. Ultimately, the promoter sequences of thepresent invention are directly incorporated into suitable planttransformation expression vectors comprising the 5′ regulatory sequencesoperatively linked to selectable markers and genes of interest,transformed into plants and the plants analyzed for the desiredexpression profile conferred by the 5′ regulatory sequences.

[0123] Those of skill in the art are aware of the vectors and suitablefor plant transformation. Suitable vectors would include but are notlimited to disarmed Ti-plasmids for Agrobacterium-mediated methods.These vectors can contain a resistance marker, 1 or more T-DNA borders,or 1 or more T-DNAs and origins of replication for E. coli andAgrobacterium along with one or more genes of interest and associatedregulatory regions. Those of skill in the art are aware that forAgrobacterium-mediated approaches a number of strains and methods areavailable. Such strains would include but are not limited toAgrobacterium strains C58, LBA4404, EHA101 and EHA105. Particularlypreferred strains are Agrobacterium tumefaciens strains. Other DNAdelivery systems for plant transformation are also known to those ofskill in the art and include but is not limited to particle bombardmentof selected plant tissues.

[0124] Exemplary nucleic acids that may be introduced by the methodsencompassed by the present invention include, for example, DNA sequencesor genes from another species, or even genes or sequences that originatewith or are present in the same species, but are incorporated intorecipient cells by genetic engineering methods rather than classicalreproduction or breeding techniques. However, the term exogenous, isalso intended to refer to genes that are not normally present in thecell being transformed, or perhaps simply not present in the form,structure, etc., as found in the transforming DNA segment or gene, orgenes that are normally present yet that one desires, e.g., to haveover-expressed. Thus, the term “exogenous” gene or DNA is intended torefer to any gene or DNA segment that is introduced into a recipientcell, regardless of whether a similar gene may already be present insuch a cell. The type of DNA included in the exogenous DNA can includeDNA that is already present in the plant cell, DNA from another plant,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

[0125] The plant transformation vectors containing the promotersequences of the present invention may be introduced into plants by anyplant transformation method. Several methods are available forintroducing DNA sequences into plant cells and are well known in theart. Suitable methods include but are not limited to bacterialinfection, binary bacterial artificial chromosome vectors, directdelivery of DNA (e.g. via PEG-mediated transformation,desiccation/inhibition-mediated DNA uptake, electroporation, agitationwith silicon carbide fibers, and acceleration of DNA coated particles(reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol., 42:205, 1991).

[0126] Methods for specifically transforming dicots primarily useAgrobacterium tumefaciens. For example, transgenic plants reportedinclude, but are not limited to, cotton (U.S. Pat. No. 5,004,863; U.S.Pat. No. 5,159,135; U.S. Pat. No. 5,518,908, WO 97/43430), soybean (U.S.Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et al.,Bio/Technology, 6:923, 1988; Christou et al., Plant Physiol., 87:671,1988); Brassica (U.S. Pat. No. 5,463,174), and peanut (Cheng et al.,Plant Cell Rep., 15: 653, 1996).

[0127] Similar methods have been reported in the transformation ofmonocots. Transformation and plant regeneration using these methods havebeen described for a number of crops including, but not limited to,asparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad.Sci. U.S.A., 84: 5345, 1987); barley (Hordeum vulgarae; Wan and Lemaux,Plant Physiol., 104: 37, 1994); maize (Zea mays; Rhodes, C. A., et al.,Science, 240: 204, 1988; Gordon-Kamm, et al., Plant Cell, 2: 603, 1990;Fromm, et al., Bio/Technology, 8: 833, 1990; Koziel, et al.,Bio/Technology, 11: 194, 1993); oats (Avena sativa; Somers, et al.,Bio/Technology, 10: 1589, 1992); orchardgrass (Dactylis glomerata; Horn,et al., Plant Cell Rep., 7: 469, 1988); rice (Oryza sativa, includingindica and japonica varieties, Toriyama, et al., Bio/Technology, 6: 10,1988; Zhang, et al., Plant Cell Rep., 7: 379, 1988; Luo and Wu, PlantMol. Biol. Rep., 6: 165, 1988; Zhang and Wu, Theor. Appl. Genet., 76:835, 1988; Christou, et al., Bio/Technology, 9: 957, 1991); sorghum(Sorghum bicolor; Casas, A. M., et al., Proc. Natl. Acad. Sci. U.S.A.,90: 11212, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J.,2: 409, 1992); tall fescue (Festuca arundinacea; Wang, Z.Y. et al.,Bio/Technology, 10: 691, 1992); turfgrass (Agrostis palustris; Zhong etal., Plant Cell Rep., 13: 1, 1993); wheat (Triticum aestivum; Vasil etal., Bio/Technology, 10: 667, 1992; Weeks T., et al., Plant Physiol.,102: 1077, 1993; Becker, et al., Plant, J. 5: 299, 1994), and alfalfa(Masoud, S. A., et al., Transgen. Res., 5: 313, 1996). It is apparent tothose of skill in the art that a number of transformation methodologiescan be used and modified for production of stable transgenic plants fromany number of target crops of interest.

[0128] The transformed plants are analyzed for the presence of the genesof interest and the expression level and/or profile conferred by thepromoter sequences of the present invention. Those of skill in the artare aware of the numerous methods available for the analysis oftransformed plants. A variety of methods are used to assess geneexpression and determine if the introduced gene(s) is integrated,functioning properly, and inherited as expected. For the presentinvention the promoters can be evaluated by determining the expressionlevels of genes to which the promoters are operatively linked. Apreliminary assessment of promoter function can be determined by atransient assay method using reporter genes, but a more definitivepromoter assessment can be determined from the analysis of stableplants. Methods for plant analysis include but are not limited toSouthern blots or northern blots, PCR-based approaches, biochemicalanalyses, phenotypic screening methods, field evaluations, andimmunodiagnostic assays.

[0129] The methods of the present invention including, but not limitedto cDNA library preparation, genomic library preparation, sequencing,sequence analyses, PCR technologies, vector construction, transientassays, and plant transformation methods are well known to those ofskill in the art and are carried out using standard techniques ormodifications thereof.

[0130] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention, therefore all matter set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

EXAMPLES Example 1

[0131] Plant Material, DNA Isolation and cDNA Library Construction

[0132] A number of tissues and plant developmental stages are selectedfor preparation of the corn libraries. Those of skill in the art areaware of the variations in tissue selection and preparation that occurfrom one tissue sampler to the next. The following are the conditionsfor the target libraries:

[0133] Seeds are planted at a depth of about 3 cm in soil into 2″-3″pots containing Metro Mix 200 growing medium and transplanted intolarger 10″ pots containing the same soil after 2-3 weeks. Peters15-16-17 fertilizer is applied about 3 times per week aftertransplanting, at a strength of 150 ppm N 2-3 times during the life ofthe plant from transplanting to flowering. A total of about 900 mg Fe isadded to each pot. Corn plants are grown in the greenhouse in 15 hrday/9 hr night cycles. The daytime temperature is 80° F. and the nighttemperature is 70° F. Lighting is provided by 1000W sodium vapor lamps.

[0134] Tissue Isolation

[0135] A root cDNA library is generated from corn (Zea mays) root tissueat the V8 plant developmental stage. The root tissue is collected whenthe corn plant is at the 8-leaf stage. The root system is cut from theplant and rinsed with water to remove the soil. The tissue is frozen inliquid nitrogen and the harvested tissue is stored at −80° C. until theRNA is prepared. cDNA synthesis is initiated using a NotI-oligo(dT)primer. Double-stranded cDNA is blunted, ligated to SalI adaptors,digested with NotI, size-selected, and cloned into the NotI and Sal1sites of the pSPORT vector (LTI). cDNA synthesis is initiated using aNotI-oligo(dT) primer. Double-stranded cDNA is blunted, ligated to EcoRIadaptors, digested with NotI, size-selected, and cloned into the NotIand EcoRI sites of the pINCY vector (Incyte Genomics, Palo Alto,Calif.).

[0136] The corn undeveloped leaf cDNA library is generated from youngcorn plants. The tissue is collected when the corn plant is at a 6-leafdevelopmental stage. The second youngest leaf that is at the base of theapical leaf of the V6 corn plant is cut at the base and immediatelytransferred to liquid nitrogen containers and the leaves are crushed.The harvested tissue is stored at −80° C. until the RNA is prepared.

[0137] For preparation of the cDNA libraries, the RNA is purified usingTrizol reagent available from Life Technologies (Gaithersburg, Md.)essentially as recommended by the manufacturer. Poly A+RNA (mRNA) ispurified using magnetic oligo dT beads essentially as recommended by themanufacturer (Dynabeads, Dynal Corporation, Lake Success, N.Y.).

[0138] Construction of cDNA libraries is well-known in the art and anumber of cloning strategies exist. A number of cDNA libraryconstruction kits are commercially available. The Superscript™ PlasmidSystem for cDNA synthesis and Plasmid Cloning (Gibco BRL, LifeTechnologies, Gaithersburg, Md.) is used, following the conditionssuggested by the manufacturer.

[0139] The cDNA libraries are plated on LB agar containing theappropriate antibiotics for selection and incubated at 37° C. for asufficient time to allow the growth of individual colonies. Singlecolonies are individually placed in each well of a 96-well microtiterplate containing LB liquid including selective antibiotics. The platesare incubated overnight at approximately 37° C. with gentle shaking topromote growth of the cultures. The plasmid DNA is isolated from eachclone using Qiaprep Plasmid Isolation kits, using the conditionsrecommended by the manufacturer (Qiagen Inc., Santa Clara, Calif.).

[0140] Template plasmid DNA clones are used for subsequent sequencing.For sequencing, the ABI PRISM dRhodamine Terminator Cycle SequencingReady Reaction Kit with AmpliTaq® DNA Polymerase, FS, is used (PEApplied Biosystems, Foster City, Calif.).

Example 2

[0141] Promoter Identification

[0142] A database of EST sequences derived from the cDNA librariesprepared from various corn tissues is used to identify the promotercandidates for expression of operably linked DNA sequences in multipletissues. The sequences are also used as query sequences against GenBankdatabases that contain previously identified and annotated sequences andsearched for regions of homology using BLAST programs. For example, thetranslation product of the EST sequence (Zm.700102320EST, SEQ ID NO: 12)starting at position 100 of SEQ ID NO: 12 shows homology tonicotianamine synthase from Hordeum vulgare (Higuchi et al., 1999. PlantPhysiol. 119:471-480, herein incorporated by reference in its entirety)and Oryza sativa (Higuchi et al., 2001. Plant J. 25:159-167, hereinincorporated by reference in its entirety). The translation of the ESTsequence is used to identify the P-Zm.700203408 genomic DNA fragmentfrom Zea mays and this polynucleotide has homology to metallothioneinfrom Zea mays (Chevalier et al. 1995. Plant Mol Biol 28:473-485), andthe translation of the EST sequence is used to identify theP-Zm.700204518 genomic DNA fragment from Zea mays and thispolynucleotide has homology to the pathogenesis-related protein fromSorghum bicolor (Lo et al. 1998. Plant Physiol. 116:979-989). Theselection of expressed sequence tags (ESTs) for subsequent promoterisolation is based on the presence of one or more sequences among therepresentative ESTs from a random sampling of an individual cDNA libraryor collection of cDNA libraries. The Zm.700102320EST is used to identifya genomic DNA fragment from Zea mays that is the promoter polynucleotideof the present invention, P-Zm.700102320.

[0143] cDNA libraries can be made from any plant species. Those of skillin the art can use the Zm.700102320EST sequence of the present invention(SEQ ID NO: 12) to design a mixture of synthetic polynucleotides ofdegenerate DNA polynucleotide sequence to the translation product of theZm.700102320EST for use as primers or probes to identify and isolateassociated DNA molecules from a plant genomic DNA library, e.g., genomicDNA libraries made from DNA isolated from rice (Oryza sativa), barley(Hordeum vulgare), corn (Zea mays) or from any plant species. Theseprimers in combination with a random primer can be used to produce anamplicon in a PCR method from corn or heterologous genomic DNAlibraries. The amplicon can be inserted into a plant expressionconstruct and tested for the capability to enhance transgene expressionin root cells.

Example 3

[0144] Genomic Library Construction, PCR Amplification and PromoterIsolation

[0145] For a genomic library, corn DNA from maize hybrid(Fr27rhm×FrMo17rhm, Illinois Foundation Seed Co.) is isolated by a CsClpurification protocol according to Ausubel et al., 1992, by a CTABpurification method (Rogers et al., Plant Mol. Biol., 5:69, 1988) or asimilar DNA isolation method suitable for the isolation of plant DNA.Reagents are available commercially (see, for example Sigma ChemicalCo., St. Louis, Mo.). The libraries are prepared according tomanufacturer instructions (GENOME WALKER, a trademark of CLONTECHLaboratories, Inc, Palo Alto, Calif.). In separate reactions, genomicDNA is subjected to restriction enzyme digestion overnight at 37° C.with the following blunt-end endonucleases: EcoRV, ScaI, DraI, PvuII, orStuI (CLONTECH Laboratories, Inc. Palo Alto, Calif.). The reactionmixtures are extracted with phenol:chloroform, ethanol precipitated, andresuspended in Tris-EDTA buffer. The purified blunt-ended genomic DNAfragments are then ligated to the GenomeWalker™ adaptors and ligation ofthe resulting DNA fragments to adaptors are done according tomanufacturer protocol. The GenomeWalker™ sublibraries are aliquoted andstored at −20° C.

[0146] Genomic DNA ligated to the GenomeWalker™ adaptor from the genomiclibrary is subjected to PCR amplification in separate reactions withgene-specific primers (GSP1) of the primary reaction, GSP1.HH (SEQ IDNO: 1) and adaptor primer 1 (AP1) SEQ ID NO: 4; GSP3.G (SEQID NO: 6.)and adaptor primer 1 (API) SEQ ID NO: 4; and GSP5.P (SEQ ID NO: 9) andadaptor primer 1 (AP1) SEQ ID NO: 4. A diluted (1:50) aliquot of each ofthe primary PCR reactions is used as the input DNA for a nested round ofPCR amplification in separate reactions with gene-specific primers(GSP2) of the secondary reaction, GSP2.II (SEQ ID NO: 2), or GSP4.H (SEQID NO: 7) or GSP6.Q (SEQ ID NO: 10) and adaptor primer 2 (AP2) SEQ IDNO: 5. The primers in the second PCR reaction have encorporatedBglII/NcoI endonuclease restriction sites. The annealing temperatures ofthe GenomeWalker™ primary primer (AP1) and nested primer (AP2) are 59°C. and 71° C., respectively. Generally, gene specific primers aredesigned to have the following characteristics: 26-30 nucleotides inlength, GC content of 40-60% with resulting temperatures for most of thegene specific primers in the high 60° C. range or about 70° C. The Taqpolymerase used is Amplitaq Gold™, available through Perkin-ElmerBiosystems (Branchbury, N.J.). A number of temperature cyclinginstruments and reagent kits are commercially available for performingPCR experiments and include those available from PE Biosystems (FosterCity, Calif.), Strategene (La Jolla, Calif.), and MJ Research Inc.(Watertown, Mass.). Any successful PCR conditions and methods can beused including but not limited to the modifications as described inTable 1. Following the primary PCR reaction, an aliquot is taken(10-15μl) for agarose gel analysis. Isolation of each unknown sequencerequires amplification from sub-genomic libraries and a negative control(without DNA). TABLE 1 PCR Conditions The PCR components and conditionsgenerally used are outlined below: PRIMARY PCR (Method 1) ComponentAmount/Volume required Sub-library aliquot 1 μl Gene-specific primer 1μl (100 pmol) Genome Walker ™ Adaptor primer 1 (AP1) 1 μl dNTP mix (10mM of each dNTP) 1 μl DMSO 2.5 μl (or 2-5% final concentration) 10X PCRbuffer (containing MgCl₂) 5 μl (final concentration of 1X) AmplitaqGold ™ 0.5 μl Distilled Water For final reaction volume of 50 μlReaction Conditions for Primary PCR: A. 9 minutes at 95° C. B. 94° C.for 2 seconds, 70° C. for 3 minutes; repeat 94° C./70° C. cycling fortotal of 7 times C. 94° C. for 2 seconds, 65° C. for 3 minutes; repeat94° C./65° C. cycling for total of 36 times D. 65° C. for 4 minutes as afinal extension E. 10° C. for an extended incubation NESTED PCR(secondary PCR reaction) Component Amount/Volume Required 1:50 dilutionof the primary PCR reaction 1 μl Gene-specific primer 1 μl (100 pmol)GenomeWalker ™ Adaptor primer 2 1 μl dNTP mix (10 mM of each dNTP) 1 μlDMSO 2.5 μl 10X PCR buffer (containing MgCl₂) 5 μl (final concentrationof 1X) Amplitaq Gold ™ 0.5 μl Distilled water to final reaction volumeof 50 μl Reaction Conditions for Nested PCR: A. 9 minutes at 95° C. B.94° C. for 2 seconds, 70° C. for 3 minutes; repeat 94° C./70° C. cyclingfor total of 5 times C. 94° C. for 2 seconds, 65° C. for 3 minutes;repeat 94° C./65° C. cycling for total of 24 times D. 65° C. for 4minutes as a final extension E. 10° C. for an extended incubationMODIFICATION 1: Polymerase (Expand High Fidelity, Boehringer Mannheim,IN) Primary PCR reaction step 1 95° C. 2 min step 2 94° C. 2 sec step 372° C. 3 min step 4 repeat step 2 and 3, 7 times step 5 94° C. 2 secondsstep 6 68° C. 3 minutes step 7 repeat steps 5 and 6, 36 times SecondaryPCR reaction step 1, step 2, step 3 (same as primary PCR reaction) step4 repeat step 2 and 3, 5 times step 5 and step 6 (same as primary PCRreaction) step 7 repeat step 5 and step 6, 24 times MODIFICATION 2:Polymerase (Amplitaq Gold ™, Perkin Elmer, Foster City, CA) Primary PCRreaction step 1 95° C. 10 minutes step 2 94° C. 2 seconds step 3 70° C.3 minutes step 4 repeat step 2 and 3, 7 times step 5 94° C. 2 secondsstep 6 68° C. 3 minutes step 7 repeat step 5 and step 6, 24 timesMODIFICATION 3: Polymerase enzyme is Taq (Promega Corp., Madison, WDsame cycle conditions as Modification 2 except step 1 is 2 minutes.MODIFICATION 4: Polymerase enzyme is AccuTaq (Sigma, St. Louis, MO) samecycle conditions as Modification 3. MODIFICATION 5: Polymerase is ExpandHigh Fidelity (BM) Mixture Primary PCR Secondary PCR 10X PCR buffer 2 2μl 1 μl dNTP 1 μl 1 μl adaptor primer (10 mM) 1 μl AP1 1 μl AP2 genespecific primer (10 mM) 1 μl GSP1 1 μl GSP2 Polymerase 2.5 units 3.5units template DNA GenomeWalker ™ library 1 μl 1:50 of primary PCRproduct H2O to 20 μl to 50 μl Modification 5: PCR Cycling Conditions:step 1 94° C. 1 minutes step 2 94° C. 2 seconds step 3 70° C. 3 minutesstep 4 repeat step 2 another 5 cycles step 5 94° C. 2 seconds step 6 68°C. 3 minutes step 7 repeat step 5 another 34 cycles step 8 68° C. 10minutes step 9 10° C. hold MODIFICATION 6: PCR Conditions step 1 95° C.2 minutes step 2 94° C. 30 seconds step 3 65° C. 30 seconds, decrease 1°C. each cycle for 14 cycles step 4 72° C. 3 minutes step 5 repeat 2-4,14 times step 6 50° C. step 7 72° C. step 8 repeat 6-7, 15 times

[0147] Those of skill in the art are aware of the variations in PCRconditions including choice of polymerase, cycling conditions andconcentrations of the reaction components. Examples of othermodifications to the above procedure include, but are not limited to theuse of Expand High Fidelity polymerase (Boehringer Mannheim,Indianapolis, Ind.), Taq polymerase (Promega Corp., Madison, Wis.) andslight variations in temperature as described in Table 1.

Example 4

[0148] Promoter Isolation and Cloning

[0149] The DNA fragments resulting from the nested PCR amplificationdescribed in Example 3 are isolated and gel purified. A 40 μl aliquot ofthe secondary PCR is run on an agarose gel. The DNA fragment of thesecondary PCR product is purified from the agarose gel using the BIO101Geneclean II Kit (Midwest Scientific, Valley Park, Mo.) following theconditions suggested by the manufacturer. The purified DNA is digestedwith one or more restriction endonuclease(s) to permit ligation into asuitable cloning or expression vector. The promoter fragments areincorporated into a plant expression vector by positioning the Zea maysroot promoter fragments in linkage with a reporter coding sequence byrestriction enzyme digestion and ligation using methods known in the art(Sambrook et al., 1989). A suitable plant expression cassette comprisesadditional genetic components that enhance gene expression in plantcells, e.g., with maize cells, one can use the I-Zm.Hsp70, an intron ofthe maize heat shock protein as described in U.S. Pat. Nos. 5,593,874,herein incorporated by reference in its entirety; and the T-AGRTU.nos(T-nos, T-NOS 3′), a transcription termination signal from the nopalinesynthase gene isolated from Agrobacterium tumefaciens. The purified DNAof the present invention is ligated as a BglII/Sal1 fragment into aconstruct that contains the necessary plant expression elements inoperably linkage. An aliquot of the ligation reaction is transformedinto a suitable E. coli host such as DH10B and the cells plated onselection medium (for DH10B, 100 μg/ml carbenicillin). Bacterialtransformants are selected, grown in liquid culture, and the plasmid DNAisolated using a commercially available kit such as the Qiaprep SpinMicroprep Kit (Qiagen Corp., Valencia, Calif.). Purified plasmidcontaining the predicted insert size are DNA sequenced in bothdirections using the dye terminator method and DNA primers homologous tothe Zm.Hsp70 intron sequence and homologous to the vector sequencebordering the promoter insertion site. Additional primers are preparedbased on the sequence produced from the first reaction and subsequentreactions. Restriction enzymes are available from a number ofmanufacturers (see for example, Boehringer Mannheim (Indianapolis,Ind.). The DNA sequences of the purified PCR product are identified asP-Zm.700102320 (SEQ ID NO: 3), P-700203408 (SEQ ID NO: 8) andP-700204518 (SEQ ID NO: 11).

Example 5

[0150] Plant Cell Analysis of Promoter Activity

[0151] The promoter fragments P-Zm.700102320, P-Zm.700204518 andP-Zm.700203408 are cloned into a plant expression cassette (pMON194690,FIG. 1) that expresses the Ec.GUS:1, a variant polynucleotide sequenceof the reporter gene encoding β-glucuronidase for enhanced expression inplant cells (GUS, U.S. Pat. No. 5,859,347, herein incorporated byreference in its entirety). The resulting plant expression cassettes arecontained in plasmids pMON33336 (P-Zm.700102320, FIG. 2), pMON33292(P-Zm.700203408, FIG. 3), and pMON33333 (P-Zm.700204518, FIG. 4). Thepromoter DNA fragments of the present invention can replace the promoterelements of any plant expression construct to determine the promoteractivity of the DNA fragments. Reporter coding sequences other than GUScan be used, such as luciferase (LUX) and green florescent protein(GFP), or any coding sequence for which an immunological or biochemicalassay is available to detect or quantify the protein expressed.

[0152] The purified plasmid DNA of pMON33336, pMON33292, and pMON33333are tested in a corn root and leaf protoplast electroporation assay forGUS activity relative to a CaMV 35S-GUS expression cassette (pMON19469)used as a control. A number of assay methods for GUS expression areavailable and known to those of skill in the art as well as methods forpreparation of leaf and root protoplasts.

[0153] A corn leaf protoplast isolation and electroporation protocol isfollowed essentially as described by Sheen, (Plant Cell 3:225-245, 1991,herein incorporated by reference in its entirely) with the followingmodifications: The seed used is FR27rhm×FRMo17rhm (Ilinois FoundationSeeds, Champaign, Ill.). The seed is surface sterilized for 2 minutes in95% ethanol, rinsed twice with sterile water, 30 minutes in 50% bleach(Clorox) plus 2 drops of Tween-20, three rinses in sterile waterfollowed by a 5 minute soak in benlate/captan solution to prevent fungalgrowth. The seeds are germinated in phytotrays containing 100milliliters ½ MS media (2.2 g/L MS salts, 0.25% gelrite), 8 seeds perphytotray. The seeds are grown 5 days at 26° C. in 16/8 hour day/nightphotoperiod and 7 days in the dark at 28° C. The second leaf from eachplant is sliced longitudinally using Feather no. 11 surgical blades.Digestion time is two hours and 10 minutes in the light at 26° C. Afterdigestion, the plates are swirled two times at 80-100 rpm for 20 secondseach and the protoplast/enzyme solution is pipetted through a 190 μmtissue collector. Protoplasts are counted using a hemacytometer countingonly protoplasts that are intact and circular. Ten to fifty microgramsof DNA containing the vector of interest is added per cuvette. Finalprotoplast densities at electroporation range from 3×106/ml to4.5×106/ml. Electroporations are performed in the light using Bio-radGene pulser cuvettes (Bio/Rad Hercules, Calif.) with a 0.4 cm gap and amaximum volume of 0.8 ml at 125 μFarads capacitance and 260 volts. Theprotoplasts are incubated on ice after resuspension in electroporationbuffer and are kept on ice in cuvettes until 10 minutes afterelectroporation. The protoplasts are kept at room temperature for tenminutes before adding 7 milliliters of protoplast growth media. Theprotoplast culture media has been described (Fromm et al., Methods inEnzymology 153, 351-366, 1987). Culture plates are layered with growthmedia and 1.5% SeaPlaque agarose (FMC BioProducts, Rockland, Me.) toprevent protoplast loss. Samples are cultured in the light at 26° C.,16/8 day/night cycle, until harvested for the assay (typically 18-22hours after electroporation). Samples are pipetted from the petri platesto 15 ml centrifuge tubes and harvested by centrifugation at 800-1000rpm. The supernatant is removed and samples are assayed immediately forthe gene of interest. Samples can also be frozen for later analysis.

[0154] Corn root protoplast isolation is performed using modificationsto the protocol of Sheen et al. (The Plant Cell Vol. 3, 225-245, 1991).Seeds (FR27×FRMo17, Illinois Foundation Seeds) are sterilized in a 500ml sterile Corning storage bottle, polystyrene with a plug seal cap.Sterilization comprised covering the seeds with 95-100% ethanol for 2minutes. The seeds are then rinsed twice with sterile distilled water.Two drops of Tween 20 are added to the bottle, and the seeds are thencovered with 50% Clorox® bleach (sodium hypochlorite) and allowed to sitfor 30 minutes. The seeds are then rinsed four times with steriledistilled water, treated with 0.25 tsp Orthocide® (Captan GardenFungicide, Chevron Chemical Co., San Ramon, Calif.) and 1 tsp Benlate®(50% benomyl, 50% inert ingredients; E.I. du Pont de Nemours and CompanyAgricultural Products, Wilmington, Del.), covered with sterile distilledwater, and allowed to sit for 5 minutes.

[0155] Seedlings are germinated, 8 per Phytatray II™, on ½ MS medium(2.2 g/L MS Basal Salts (M-5524), 2.5 g/L Phytagel™) at approximately 80mL per Phytatray II™. The seedlings are germinated embryo side down for4 days in the light (incubator at 26° C. with a 16 hr day/8 hr nightcycle under cool white fluorescent bulbs, 10-25 μE).

[0156] After germination, the seedlings are pulled sideways from thephytatrays and then upwards to minimize the amount of media removed withthe roots. Large main roots are removed by cutting at the base of theseed with a blade. The tissue is then wounded with a triple-bladedscalpel (three scalpels bond together in parallel) at an angle of 45degrees to the direction of root length. The wounds are madeperpendicular to the direction of growth and the segments about 2-4 mmin length.

[0157] Six to seven grams of wounded material is placed in a deep petridish (100×25mm) that contains 40 ml of enzyme mix (1.5% cellulase YC,0.1% Pectolyase, 0.7 M mannitol, 10 mM MES(2-[N-morpholino]ethanesulfonic acid), 1 mM CaCl₂, 1 mM MgCl₂, 0.1%bovine serum albumin (BSA), and 17 mM Beta-mercaptoethanol; pH 5.7). Thepetri dish with enzyme and root tissue is then vacuum infiltrated for 30seconds.

[0158] Digestion is performed in the light (cool white fluorescentbulbs, 10-25 μE) for 135 minutes at 50 rpm on an Orbit™ platform shakerat 26° C. After digestion, plates are swirled by hand at about 100 rpmfor 50 seconds to release protoplasts from the tissue mass. Protoplastsare separated out by straining the enzyme mix through a sievingassembly. The sieving assembly consists of one collector with a widemesh (Bellco Glass, Inc. Cat #1985-00030) that is placed inside ofanother collector with a 190 μm sieve. The sieved protoplasts aretransferred to a 50 mL conical bottom centrifuge tube, and pelleted bycentrifugation at 200×g for 8 minutes. The pellet is resuspended in 10mL of rinse media (0.7 M mannitol, 4 mM MES (pH 5.7), pH 5.7) andcentrifuged again at 200×g for 8 minutes.

[0159] The pellet is then resuspended in 10 mL of electroporation buffer(0.7 M mannitol, 4 mM MES, 1.0 mM Beta-mercaptoethanol, 25 mM KCl, pH5.7), the protoplasts are counted with a Hausser ScientificBright-Line™_0 hemacytometer. The protoplasts are then pelleted againand resuspended in electroporation buffer at a density of 0.5×10⁶cells/mL.

[0160] In preparation for transfection, 750 μl of protoplasts at 0.5×10⁶cells/mL are added to each BioRad Gene Pulser® cuvette (0.4 cm gap)followed by the addition of DNA. Transfection is performed byelectroporation at 125 μF and 250 V on a BioRad Gene Pulser™ Model No.1652076, BioRad Capacitance Extender Model No. 1652087. Prior to andpost transfection the cuvettes are placed on ice for 10 minutes. Theprotoplasts and DNA are mixed by inverting the cuvettes twiceimmediately prior to electroporation.

[0161] After transfection, protoplasts are poured into agarose layeredplates (MS Fromm+0.7 M mannitol+15 g/L SeaPlaque® agarose (FMC®Bioproducts)) in 3.5 mL of MS Fromm+0.7 M mannitol (4.4 g/L MS salts(Gibco, 500-1117EH), 1 mL/L 1000×vitamins (1.3 g/L nicotinic acid, 250mg/L thiamine HCl, 250 mg/L pyridoxine HCl, 250 mg/L calciumpanthothenate), 20 g/L sucrose, 2 mg/L 2,4-D, 0.1 g/L inositol(myo-inositol), 0.13 g/L asparagine, 127 g/L mannitol) and culturedovernight. All chemicals used are obtained from Sigma Chemical Company,St. Louis, Mo., except as indicated. This overnight culture is performedin an incubator at 26° C. with a 16 hr day/8 hr night cycle utilizingcool white fluorescent bulbs, 10-25 μE.

[0162] Protoplasts are harvested after one day; culture time is 18-22hr. Protoplasts are removed from the plate using a 10 mL serologicalpipette, with care taken not to draw up the agarose layering.Protoplasts are then put in 15 mL conical bottom centrifuge tubes andcentrifuged at 200×g for 8 minutes. The supernatant is removed and thepellets are placed immediately on dry ice. All pellets are then storedin a −80° C. freezer until assayed.

[0163] The expression of GUS by the corn root promoters is measured as apercentage of the activity observed when pMON19469 is set to 100. Thetest constructs are electroporated into corn leaf and root protoplastsand replicated 2 or 4 times as shown in Table 2. The assay is performedby the MUG method that provides a quantitative analysis of the GUSexpression in the transgenic plant cells. Total protein is extractedfrom each sample, measured and concentration adjusted such that eachsample contains the same amount of total protein. Total protein isassayed using Bio-Rad Protein Assay kit. Serial dilutions of BSA proteinfrom 0.05 mg/ml to 0.5 mg/ml are used for the standard curve. The MUGassay uses 500 μl of GUS extraction buffer added to the tissues, andtissues are ground with a teflon pestle in 1.5 ml eppendorf tube andcentrifuged at 10K RPM for 5 minutes at 4 ° C. (Beckman GS-15R). Fourhundred μl of supernatant is transferred to a fresh 96-deep well plate.The extracts are frozen on dry ice, then stored at −80 ° C. until use.The MUG assay consisted of generating a standard curve of activity witha serial dilution of 4-methyl umbelliferone (Sigma Chemical CoCat#M1381, St Louis, Mo.) from 31.2 ρmoles to 2000 ρmoles. Five μl ofeach extract is added to a flat bottom 96-well plate (Falcon #3872, BDBiosciences) in duplicate after the plate is read for blanking thebackground. Two hundred μl of GUS assay solution (0.1M KPO₄ pH7.8, 1.0mM EDTA, 5% glycerol, 10.0 mM DTT, 2 mM 4-methyl umbelliferylglucuronide, Fluka #69602) is added to each well and mixed with thesamples by pipetting. The Plate is read kinetically on a F-max(Molecular Devices, Sunnyvale Calif.) at 37° C. with the filter pair:excitation-355/emission-460. A typical read consists of 21 readings at 3minute intervals. GUS activity (pmol/min/mg protein) is calculated baseon MUG results and protein results of each sample. 1.5 μl of extracts isadded to flat bottom 96-well plate (Falcon) in duplicate. 200 ul ofdiluted dye reagent is added and mixed with the samples. The absorbanceat 595 nm is measured in Spectromax 250 (Molecular Devices, SunnyvaleCalif.) at room temperature after 5 minutes incubation at roomtemperature.

[0164] A control plasmid (Luc plasmid) containing the plant expressioncassette for CaMV.35S/Luc/T-nos is co-electroporated to standardize theelectroporation experiments. The same amount of the Luc plasmid is addedto each solution with the test constructs and electoporated. A sample ofthe electroporated cells are assayed for luciferase activity after eachtest. Quantitative luciferase assays is performed as follows: 50 μl ofextract is added to a cuvette containing 0.2 mls of 25 mM Tricine pH7.8,15 mM MgCl₂, 5 mM ATP, and 0.5 mg/ml BSA. 0.5 mM luciferin substrate isautomatically dispensed by the luminometer (Berthold Bioluminat LB9500C)and the peak luminescence measured during a 10 second count at 25° C.Three to ten reactions are run per sample. The values shown in table 2have been normalized to have equivalent Luc expression in each sample.TABLE 2 Corn protoplast analysis of corn root promoter sequences drivingexpression of GUS: 1 Root Proto- Leaf Promoter DNA pMON plast protoplastRoot/leaf ratio 1 CaMV 35S 19469 100 100 1 2 P-Zm.700203408 33292 70, 9928, 32, 84.5/39.2 = 2.1 56, 41 3 P-Zm.700204518 33333 36, 28, 19, 18,39.5/17.7 = 2.2 48, 46 17, 17 4 P-Zm.700102320 33336 23, 44 2, 2, 2, 2   33.5/2 = 16.7

[0165] The analysis of the root promoter DNA sequences shows thatP-Zm.700102320 is highly enhanced for expression in root cells relativeto leaf cells with a ratio of 16.7. Although, the relative expressionlevel of GUS driving by the P-Zm.700102320 promoter in root cells isabout one third of that observed for the CaMV 35S promoter, theextremely low leaf protoplast expression makes this promoters desirablefor use to direct differential expression in a transgenic plant.

Example 6

[0166] DNA constructs are made for plant transformation by the additionof a selectable marker expression cassette. The promoter DNA fragmentsof P-Zm.700102320 (SEQ ID NO: 3), P-Zm.700203408 (SEQ ID NO: 8) andP-Zm.700204518 (SEQ ID NO: 11) are ligated into a plant expressioncassette containing the genetic elements for expression of GUS inmonocot plants. The construct pMON46152 (FIG. 6) is constructed to linkthe P-Zm.700102320/1-Hsp70/Ec.GUS:1/T-nos together in an expressioncassette and to contain the P-CaMV.35S/nptII/T-nos expression cassettein the same T-DNA for selection of transgenic corn plants onparamomycin. pMON46154 (FIG. 7) and pMON46155 (FIG. 8) are constructedin the same manner as pMON46152. As a comparative expression cassette,pMON18365 containing the same GUS reporter gene, is transformed in thesame experiment with the root promoter constructs.

[0167] Transgenic corn plants can be produced by an Agrobacteriummediated transformation method using the DNA constructs of the presentinvention. DNA constructs useful in plant transformation methodsmediated by Agrobacterium contain a binary construct that has one ormore Agrobacterium Ti plasmid border elements, left border (LB), RightBorder (RB), plasmid maintenance elements (ORI-322, ori-V, ROP),bacteria selectable marker genes, e.g., aad, providing resistance tospectinomycin and streptomycin (SPC/STR). A disarmed Agrobacteriumstrain C58 (ABI) harboring a binary vector of the present invention isused for all the experiments. The DNA construct is transferred intoAgrobacterium by a triparental mating method (Ditta et al., Proc. Natl.Acad. Sci. 77:7347-7351). Liquid cultures of Agrobacterium are initiatedfrom glycerol stocks or from a freshly streaked plate and grownovernight at 26° C.-28° C. with shaking (approximately 150 rpm) tomid-log growth phase in liquid LB medium, pH 7.0 containing 50 mg/lkanamycin, 50 mg/l streptomycin and spectinomycin and 25 mg/lchloramphenicol with 200 μM acetosyringone (AS). The Agrobacterium cellsare resuspended in the inoculation medium (liquid CM4C) and the densityis adjusted to OD₆₆₀ of 1. Corn embryos are dissected directly into theAgrobacterium solution with OD around 1.0 in ½ MSPL plus 200 μMacetosyringone and let sit for 5 to 10 minutes. The solution is decantedinto the center of the co-culture plates and the Agrobacterium liquid isremoved. Embryos are placed scutellum side up. Co-culture for 1 day at23° C. in the dark. Before placing on selection, embryos are placed ondelay culture (MS basal media (Murashige et al., Physiol. Plant15:473-497, 1962) containing 0.5 mg/L 2,4-D, 2.2 mg/L picloram, 500 mg/Lcarbenicillin, 20 μM silver nitrate) for 3 days. Embryos are then placedon selection media (MS basal media containing 0.5 mg/L 2,4-D, 2.2 mg/Lpicloram, 500 mg/L carbenicillin, 20 μM silver nitrate, and 50 mg/Lparomomycin) for 2 weeks. After 2 weeks, all visible coleoptiles areremoved and embryos are transferred to a second regeneration media (MSbasal media containing 0.5 mg/L 2,4-D, 2.2 mg/L picloram, 500 mg/Lcarbenicillin, 20 μM silver nitrate, and 100 mg/L paromomycin) for 2weeks. The embryos are subcultured on the same medium 2 additional timeswith a 2-week transfer cycle. All previous steps from delay throughselection occur at 27° C. in the dark. Callus events consisting ofhealthy type I tissue are transferred to MS basal medium containing 6BAfor 5 to 7 days in the light at 27° C. After the BA pulse, the eventsare transferred to MSOD plates with 100 mg/L paromomycin for 4 to 5weeks. As plants emerged, they are transferred to a covered containercontaining the same medium for rooting. Rooted plants are transferred tosoil in pots.

Example 7

[0168] Transgenic corn plants transformed with the DNA constructspMON18365 (FIG. 5), pMON46152 (FIG. 6), pMON46154 (FIG. 7) and pMON46155(FIG. 8) are growth in pots in a greenhouse. These plants are assayedfor GUS expression using the MUG assay as described in the analysis ofthe protoplasts (EXAMPLE 5). Tissue samples are collected from thenewest emerged leaf (referred herein as YL, the young leaf) at a growthstage later than V8, the fourth surviving leaf from the plant base V4,and the root. The results of the GUS analysis of these lines and tissuesas illustrated in FIG. 9. For pMON18365, the CaMV 35S promoter isdriving the expression of the GUS gene that contains the potato LS1intron (I-St.LS1), sixteen transgenic lines are assayed for expressionof GUS expressed in pmol/MU/min/mg, the young leaf (YL) samples show amean of 2,225,878, the V4 leaf samples show a mean of 2,954,229, and theroot samples show a mean of 3,517,380. For pMON46152, the P-Zm.700102320promoter is driving the expression of the GUS gene, seventeen transgeniclines are assayed for expression of GUS expressed in pmol/MU/min/mg, theyoung leaf (YL) samples show a mean of 175,802, the V4 leaf samples showa mean of 70,642, and the root samples show a mean of 2,542,226. ForpMON46154, the P-Zm.700203408 promoter is driving the expression of theGUS gene, fifteen transgenic lines are assayed for expression of GUSexpressed in pmol/MU/min/mg, the young leaf (YL) samples show a mean of1,148,794, the V4 leaf samples show a mean of 1,559,493, and the rootsamples show a mean of 3,210,164. For pMON46155, the P-Zm.700204518promoter is driving the expression of the GUS gene, fifteen transgeniclines are assayed for expression of GUS expressed in pmol/MU/min/mg, theyoung leaf (YL) samples show a mean of 911,996, the V4 leaf samples showa mean of 1,025,446, and the root samples show a mean of 2,056,952 (FIG.9).

[0169] The P-Zm.700102320 promoter demonstrates that in whole plants itprovides enhanced expression of a transgene product in root tissuesrelative to leaf tissues. The ratio of root/leaf expression is between14 and 36 fold enhancement. In the same experiment, other root promotersP-Zm.700203408 and P-Zm.700204518 only demonstrate root/leaf ratiosbetween 2 and 3 fold difference.

Example 8

[0170] The transgenic corn plants containing the root enhanced promoterconstructs are analyzed for GUS expression in the F₁ generation progeny.Ten events from each construct (pMON46152, pMON46154, pMON46155 andpMON18365) and 3 plants/event are selected for analysis of geneexpression levels in various plant tissues. Tissues that are sampled inthis study include root and youngest leaf at V4 and V8 stage, and pollenand silk. A summary of the expression data is shown in FIG. 10 for thesix tissues that are quantitatively examined. The root expression forall three promoters is greater than or about the same as the controlconstruct pMON18365 that contains the CaMV 35S promoter. The promoter,(P-Zm.700102320 (pMON46152) shows the lowest level of expression in leaftissue. The promoter, P-Zm700203408 (pMON46154) demonstrates the highestoverall expression in leaves and roots, however root expression isenhanced relative to the CaMV 35S promoter although GUS expression isdetectable in pollen. P-Zm.700204518 (pMON46155) resembles theexpression pattern observed in the tissues of transgenic plantscontaining pMON46154, except the overall expression level is reduced.This construct and pMON46152 does not show any GUS expression in pollenusing the quantitative enzyme activity assay, but faint staining couldbe seen following histochemical staining.

Example 9

[0171] Promoters that enhance the expression of transgenes in root cellsand root tissues are especially useful for the expression of proteinstoxic to root pests. Of particular interest is the expression ofinsecticidal proteins toxic to the corn root worm (Diabrotica spp.).Insecticidal protein genes isolated from Bacillus thuringiensis, e.g.ET8076 (WO 0066742, herein incorporated in its entirely), cry3Bb gene(WO 9931248, herein incorporated in its entirely), ET70 (WO 0026378,herein incorporated in its entirely), and PS149B1 (Moellenbeck, et al.2001. Nature Biotech 19:668-672, herein incorporated in its entirely),have been shown to be toxic to the corn root worm. The enhancedexpression of the gene sequences encoding these proteins in the roots ofcorn plants relative to other plant cells or tissues, reduces thepotential exposure that nontarget organisms may receive of the expressedprotein in the environment.

[0172] DNA constructs that contain the P-ZM.700102320 promoter linked tothe DNA molecule encoding the CRY3Bb protein are assembled incombination with other genetic elements that contribute to higherexpression levels by enhancing the stability or translatability of thetranscribed mRNA. Some examples include, but are not limited to theexpression cassettes contained in pMON64106 (FIG. 11) the cassetteP-Zm.700102320/1-Zm.Hsp70/cry3Bb/T-Os.LacD, pMON64107 (FIG. 12) thatcontains the cassette P-Zm.700102320/I-Zm.Hsp70/cry3Bb/T-Os.Glut,pMON64108 (FIG. 13) that contains the cassetteP-Zm.700102320/1-Os.Actl/cry3Bb/T-Os.LacD, and pMON64109 (FIG. 14) thatcontains the plant expression cassetteP-Zm.700102320/1-Os.Actl/cry3Bb/T-Os.Glut. Transformation of these DNAconstructs into corn cells and analysis of the regenerated corn plantsis shown in table 3. Root and leaf tissue is assayed for the CRY3Bbprotein (ppm) by ELISA from V4 and V8 corn growth stages. The effect ofintron and 3′ UTR regulatory elements on the expression levels in eachof the tissues is measured. All of the combinations of regulatoryelements with the P-Zm.700102320 promoter demonstrate a high root toleaf ratio of the insecticidal protein.

[0173] Additional plant expression cassettes can be included in theT-DNA of the DNA construct or in a different T-DNA residing on the sameconstruct, these can include, but are not limited to the T-Os.Lacd(lactate dehydrogenase 3′ UTR, WO 200011200, herein incorporated byreference in its entirety) and the T-Os.Glut (glutelin 3′ UTR from rice,WO 200011200, herein incorporated by reference in its entirety). Theseadditional expression cassettes can include selectable marker genes,e.g. the aroA:CP4 gene (CP4 EPSPS, U.S. Pat. No. 5,633,435) encoding aglyphosate resistant enzyme and nptII encoding neomycinphosphotransferase, as well as other genes of agronomic importance.TABLE 3 Root to leaf Bt CRY3Bb protein expression (ppm) in V4 and V8corn INTRON 3′ UTR V4 root V4 leaf V4 root/leaf V8 root V8 leaf V8root/leaf pMON64106 Hsp70 Os Lacd 9.83 0.00 NA 0.17 0.00 NA pMON64107Hsp70 Os Glut 12.67 0.40 31.68 2.67 0.00 NA pMON64108 Os Act Os Lacd13.67 0.42 32.55 8.83 0.53 16.66 pMON64109 Os Act Os Glut 11.17 0.4524.82 3.67 0.82  4.48

Example 10

[0174] Identification of Cis Acting Elements

[0175] Cis acting regulatory elements necessary for proper promoterregulation can be identified by a number of means. In one method,deletion analysis is carried out to remove regions of the promoter andthe resulting promoter fragments are assayed for promoter activity. DNAfragments that are considered necessary for promoter regulation if theactivity of the truncated promoter is altered compared to the originalpromoter fragment. Through this deletion analysis, small regions of DNAcan be identified that are necessary for positive or negative regulationof transcription. Promoter sequence motifs can also be identified andnovel promoters engineered to contain these cis elements for modulatingexpression of operably linked transcribable sequences. See for exampleU.S. Pat. No. 5,223,419, herein incorporated by reference in itsentirety, U.S. Pat. No. 4,990,607 herein incorporated by reference inits entirety, and U.S. Pat. No. 5,097,025 herein incorporated byreference in its entirety.

[0176] An alternative approach is to look for similar sequences betweenpromoters with similar expression profiles. Promoters with overlappingpatterns of activity can have common regulatory mechanisms. Severalcomputer programs can be used to identify conserved, sequence motifsbetween promoters, including but not limited to MEME, SIGNAL SCAN, orGENE SCAN. These motifs can represent binding sites for transcriptionsfactors that act to regulate the promoters. Once the sequence motifs areidentified, their function can be assayed. For example, the motifsequences can be deleted from the promoter to determine if the motif isnecessary for proper promoter function. Alternatively, the cis elementmotif can be added to a minimal promoter to test whether it issufficient to activate or enhance transcription in certain plant cellsor tissues. In the present invention, a region of DNA polynucleotidesequence of SEQ ID NO: 3 from position 110-192 and a DNA polynucleotidesequence at least 90% homologous to this sequence or a motif sequence ofDNA polynucleotide sequence of SEQ ID NO: 3 from position 126-164 and aDNA polynucleotide sequence at least 90% homologous can be assayed forroot cell and tissue transcription enhancement of transgenes. Suspectednegative regulatory elements can be tested for sufficiency by combiningwith an active promoter molecule and testing for a reduction in promoteractivity. Some cis acting regulatory elements may require other elementsto function. Therefore, multiple elements can be tested in variouscombinations by any number of methods known to those of skill in theart.

[0177] Once functional promoter elements have been identified, promoterelements can be modified at the nucleotide level to affect proteinbinding. The modifications can cause either higher or lower affinitybinding that would affect the level of transcription from that promoter.

[0178] Promoter elements can act additively or synergistically to affectpromoter activity. In this regard, promoter elements from different 5′regulatory regions can be placed in tandem to obtain a promoter with adifferent spectrum of activity or different expression profile.Accordingly, combinations of promoter elements from heterologous sourcesor duplication of similar elements or the same element can confer ahigher level of expression of operably linked transcribable sequences.For example, a promoter element can be multimerized to increase levelsof expression specifically in the pattern affected by that promoterelement. Novel cis acting elements can be combined to create chimericDNA molecules with promoter activity. The cis elements of the promoterDNA molecule of the present invention can be combined with otherpromoter DNA molecules that may be deficient in expression in root cellsand root tissues to enhance their activity in these cells and tissues,e.g. various tissue specific elements of the CaMV 35S promoter (U.S.Pat. No. 5,097,025, and U.S. Pat. No. 5,110,732, herein incorporated byreference in their entirety).

[0179] Additionally, the cis elements of the present invention can becombined with other known root cell and root tissue cis elements tocreate a novel promoter DNA molecule with enhanced root expression, e.g.the root expression element AS-1 of the CaMV 35S promoter (U.S. Pat. No.5,023,179, herein incorporated by reference in its entirety).

[0180] The technical methods needed for constructing chimeric DNAmolecules in expression vectors containing the novel engineered 5′regulatory elements are known to those of skill in the art. Theengineered promoters are tested in expression vectors and testedtransiently by operably linking the novel promoters to a suitablereporter gene, such as GUS, and testing in a transient plant assay. Thenovel promoters are operably linked to one or more genes of interest andincorporated into a plant transformation vector along with one or moreadditional regulatory elements and transformed into a target plant ofinterest by a suitable DNA delivery system. The stably transformedplants and subsequent progeny are evaluated by any number of molecular,immunodiagnostic, biochemical, phenotypic, or field methods suitable forassessing the desired agronomic characteristic(s).

[0181] Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

[0182] All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application is specifically andindividually indicated to be incorporated by reference.

1 12 1 23 DNA Zea mays 1 tcagcgacgg cagcttggcg atg 23 2 38 DNA Zea mays2 gcctccatgg tagatctggc tgctacggtt gcagttgg 38 3 677 DNA Zea mays 3aaatatcgga atattagcat gtcaacttgc actctctaag gctcctttgg aaagcaggat 60tttagaaaaa aaaatcatat aaatttttta catgaatcag tttattttcg gattatgaaa 120tattttctca taacagtata acacatattt tgtatataag ttattatgtt attatatata 180accgttgcaa cgtacgggca ttcacctagt aaagaaagaa gattaattat tctctggtgg 240agattgtgcc cgagcccgaa ggtcatgata tggacgttgc aaacccactt cacgagggga 300caaaaaagaa atagggttac cactttcatc agttaaaggg cgtgacatgg acgtgttgaa 360gatccggcac attccctgcg aaatatacac gtcatggtac taacgaggca tgaaactggc 420cacatggcca tggacgcgtg aagcgtgcca tgcattggac atgcggcatc cgaacttctg 480aagatcatat cagagagaca ctgatgtacg aactgccgta acattctatt ctatatatac 540cctcagtccc tgttccagtt ctcgttaagc tagcagcacc aagttgtcga acacttgcct 600gctcttgagc tcgatcaagc tatcatcagc tgcgtcttgc gcacagcaac agcttcccaa 660ctgcaaccgt agcagcc 677 4 22 DNA Artificial Sequence misc_feature(1)..(22) fully synthetic sequence 4 gtaatacgac tcactatagg gc 22 5 19DNA Artificial Sequence misc_feature (1)..(19) fully synthetic sequence5 actatagggc acgcgtcct 19 6 20 DNA Zea mays 6 gccgcacttg cagcttgagc 20 741 DNA Zea mays 7 gatcgatccc atggtagatc ttgtggtgct aaagaagctc g 41 81027 DNA Zea mays 8 cctccatata tgattgtcgt cgggcccata acagcatctcctccaccagt ttattgtaag 60 aataaattaa gtagagatat ttgtcgtcgg gcagaagaaacttggacaag aagaagaagc 120 aagctaggcc aatttcttgc cggcaacagg aagatagtggcctctagttt atatatcggc 180 gtgatgatga tgctcctagc tagaaatgag agaagaaaaacggacgcgtg tttggtgtgt 240 gtcaatggcg tccatccttc catcagatca gaacgatgaaaaagtcaagc acggcatgca 300 tagtatatgt atagcttgtt ttagtgtggc tttgctgagacgaatgaaag caacggcggg 360 catatttttc agtggctgta gctttcaggc tgaaagagacgtggcatgca ataattcagg 420 gaattcgtca gccaattgag gtagctagtc aacttgtacattggtgcgag caattttccg 480 cactcaggag ggctagtttg agagtccaaa aactataggagattaaagag gctaaaatcc 540 tctccttatt taattttaaa taagtagtgt atttgtattttaactcctcc aacccttccg 600 attttatggc tctcaaacta gcattcagtc taatgcatgcatgcttggct agaggtcgta 660 tggggttgtt aatagcatag ctagctacaa gttaaccgggtcttttatat ttaataagga 720 caggcaaagt attacttata aataaagaat aaagctaggacgaactggat tactaaatcg 780 aaatggacgt aatattccag gcaagaataa ttcttcgatcaggagacaag tggggcattg 840 gaccggttct tgcaagcaag agcctatggc gtggtgccacggcgcgttgc ccatacatca 900 tgcctccatc gatgatccat cctcacttgc tataaaaagaggtgtccatg gtgctcaagc 960 tcagccaagc aaataagacg acttgtttca ttgattcttcaagagatcga gcttctttag 1020 caccaca 1027 9 24 DNA Zea mays 9 tgggaggcgaccttgggcgc cagg 24 10 35 DNA Zea mays 10 tcggaggcca tggtagatctagtgatcgat cggcc 35 11 748 DNA Zea mays 11 cgatcagtct aagaatgaccagaagcaaca acgacttcag acctttagac catgacatct 60 agaagaaggt atatgcaagcaaaatacatc taaagcatct gactgactcg ttagtgctag 120 cccttcttct gaacaacttctttctaagta tatgaataag aaggtcgttt cacacaattg 180 atcgacaaaa cgatcaatatcatccacaac gaggaagcaa tccatgcaag ggcaaaagcc 240 gaataaatcg gcccaggaagtggtgcaacc aatgtcgcct actcatccgc tctaggaatg 300 tcgtgttact ttccaccagtctactcatcg atgatgtttt atcctgctaa catgtgaaaa 360 agtatgacga tgaatccgtattacacaggg gcggacgcag agggaggcaa agtgggtcat 420 agccacctca attttcatgatattttatat atcatgacgt gcagtctctt tgcaacccca 480 gccacattaa ttaatagactccaccgacga gcgacgagtg atggtaccgg ccgccggccc 540 aggccaaccc aagtggaaaaggccgacgac tcccggacgt ctcatcctca ccggacgcca 600 ccaacccccg caatctccagacgtacgagc cgcctattta aagccctcag tctgccactc 660 tcatggcaac gcaagcagaagctacaatcc taaaaccatc tgcttcagcc ttcagctagc 720 cccaagttta gtcggccgatcgatcact 748 12 298 DNA Zea mays 12 gcaccaagtt gtcgatcact tgcctgctcttgagctcgat caagctatca tcagctacag 60 cttccgatcc caactgcaac tgtagcagcgacaactgcca tggaggccca gaacgtggag 120 gttgctgccc tggtgcagaa gatcacggccctccacgccg acatcgccaa gctgccgtcg 180 ctgagcccgt cccccgacgc caacgcgctgttcaccagcc tcgtcatggc ctgcgtcccg 240 ccaaaccctg tcgacgtgac caagctcagcccggacgtcc aggggatgcg agaggagc 298

We claim:
 1. An isolated DNA molecule comprising a DNA polynucleotide atleast 90% homologous to SEQ ID NO: 3 or fragment, region, or motifthereof.
 2. A DNA molecule comprising a first DNA polynucleotide atleast 90% homologous to SEQ ID NO: 3 or fragment, region, or motifthereof, wherein said first DNA polynucleotide is operably linked to asecond DNA polynucleotide heterologous to said first polynucleotide andoperably linked to a 3′ transcription termination DNA polynucleotide. 3.The DNA molecule of claim 2, wherein said second DNA polynucleotide istranscribed into a RNA molecule that encodes a protein.
 4. The DNAmolecule of claim 2, wherein said second DNA polynucleotide istranscribed into a RNA molecule that does not encode a protein.
 5. Anisolated DNA molecule comprising a DNA polynucleotide set forth in SEQID NO: 3 or fragment, region, or motif thereof.
 6. A DNA moleculecomprising a first DNA polynucleotide set forth in SEQ ID NO: 3 orfragment, region, or motif thereof, wherein said first DNApolynucleotide is operably linked to a second DNA polynucleotideheterologous to said first DNA polynucleotide and operably linked to a3′ transcription termination DNA polynucleotide.
 7. The DNA molecule ofclaim 6, wherein said second DNA polynucleotide is transcribed into aRNA molecule that encodes a protein.
 8. The DNA molecule of claim 6,wherein said second DNA polynucleotide is transcribed into a RNAmolecule that does not encode a protein.
 9. A DNA promoterpolynucleotide isolated by a method comprising the steps of: (i)preparation of plant genomic DNA; and (ii) preparation of a DNApolynucleotide primer having sequence homology to a length of SEQ ID NO:12; and (iii) mixing said DNA polynucleotide primer and genomic DNA witha second DNA polynucleotide primer not homologous to SEQ ID NO: 12; and(iv) subjecting said mixture to a PCR condition that provides anamplicon; and (v) purifying said amplicon; (vi) inserting said ampliconinto an expression construct; and (vii) testing said expressionconstruct for expression of a reporter molecule.
 10. A DNA promoterpolynucleotide isolated by a method comprising the steps of: (i)preparation of plant genomic DNA; and (ii) preparation of a mixture ofdegenerate DNA polynucleotide primers to a length of the translationproduct of SEQ ID NO: 12; and (iii) mixing said degenerate DNApolynucleotide primers and genomic DNA with a second DNA polynucleotideprimer not homologous to SEQ ID NO: 12; and (iv) subjecting said mixtureto a PCR condition that provides an amplicon; and (v) purifying saidamplicon; (vi) inserting said amplicon into an expression construct; and(vii) testing said expression construct for expression of a reportermolecule.
 11. A DNA promoter polynucleotide isolated by a methodcomprising the steps of: (i) preparation of plant genomic DNA; and (ii)preparation of a DNA polynucleotide primer having sequence homology to a5′ length of SEQ ID NO: 3; and (iii) mixing said DNA polynucleotideprimer and genomic DNA with a second DNA polynucleotide primer havingsequence homology to a 5′ length of SEQ ID NO: 12; and (iv) subjectingsaid mixture to a PCR condition that provides an amplicon; and (v)purifying said amplicon; (vi) inserting said amplicon into an expressionconstruct; and (vii) testing said expression construct for expression ofa reporter molecule.
 12. A transgenic plant made by a method comprisingthe steps of: (i) introducing into the genome of a plant cell a DNAmolecule comprising a first DNA polynucleotide at least 90% homologousto SEQ ID NO: 3 or motif thereof, wherein said first DNA polynucleotideis operably linked to a second DNA polynucleotide heterologous to saidfirst polynucleotide and operably linked to a 3′ transcriptiontermination DNA polynucleotide; and (ii) selecting said transgenic plantcell; and (iii) regenerating said transgenic plant cell into atransgenic plant.
 13. The transgenic plant of claim 12, wherein said DNAmolecule confers disease resistance to said transgenic plant.
 14. Thetransgenic plant of claim 12, wherein said DNA molecule confers enhancedroot growth to said transgenic plant.
 15. The transgenic plant of claim12, wherein said DNA molecule confers insect resistance to saidtransgenic plant.
 16. The transgenic plant of claim 12, wherein said DNAmolecule confers herbicide tolerance to said transgenic plant.
 17. Thetransgenic plant of claim 12, wherein said DNA molecule confers stresstolerance to said transgenic plant.
 18. A seed of said transgenic plantof claim
 12. 19. The isolated DNA molecule of claim 1, wherein saidpolynucleotide is at least 90% homologous to SEQ ID NO:
 3. 20. Theisolated DNA molecule of claim 1, wherein said polynucleotide is setforth in SEQ ID NO: 3.